# Review of Cyanotoxicity Studies Based on Cell Cultures

Authors: Iliyana Sazdova; Milena Keremidarska-Markova; Mariela Chichova; Blagoy Uzunov; Georgi Nikolaev; Mitko Mladenov; Rudolf Schubert; Maya Stoyneva-Gärtner; Hristo S. Gagov
Journal: Journal of Toxicology
 (2022)

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## Abstract

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.


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## Body

## 1. 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 CyanoHABs. The 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 “sea-food”, which includes both freshwater and marine organisms [3–5]. The excretion of toxic compounds may lead to environmental disasters—water poisoning, extinction of aquatic life, and even to human death [3–5]. 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]. Thus, 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]. The 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. They allow to reveal the mechanisms of cytotoxic effects, the affected tissues, intracellular targets, and ways to minimize cytotoxicity [11]. The 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. This is caused by the high cost of these types of clinical trials and increasing resistance to the use of animals for scientific studies. Therefore, the significance and use of cell lines is gradually increasing.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 (4) the intracellular targets of CT. The 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.


## 2. Cyanotoxicity on Cell and Cell Cultures

### 2.1. Cytotoxicity of Aeruginosins (Table1)

Aeruginosin CT contains as a basic structure 2-carboxy-6-hydroxyoctahydroindol that are serine protease inhibitors [12]. They inhibit trypsin-like serine proteases and for this activity are important in the search for new anticoagulants [13].Table 1
Cytotoxicity of aeruginosins.
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.Huh7 cellsEROD assay, treatment with TNF-αAeruginosin-865A 50 and 100 µmol/LHuman hepatoma cell lineAnti-inflammatory activity by inhibition of IL-8 and TNF-α expression; induce expression of cytochrome P450 1A (CYP1A)DNA[12]HLMVECIL-8 and ICAM-1 assay upon stimulation with human tumor necrosis factorα (hTNF-α)Aeruginosin-865 0.1–100 µg/mL/18 h of 0.1 ngm/L hTNF-α stimulated cellsHuman lung microvascular endothelial cellsAnti-inflammatory activity by down-regulation of IL-8 (EC50 : 4.0 ± 1.7 mM) and intercellular adhesion molecule 1 (ICAM-1; 57.8 ± 15.5 mM)Inhibits NF-kappa B translocation to the nucleus[13]WEHI-13VARLactate dehydrogenase (LDH) cytotoxicity assayAeruginosin-865 10–200 µMMouse fibrosarcoma cellsCytotoxic effect of aeruginosin-865 at 200 µM only[14]Abbreviations: EROD – ethoxyresorufin-O-deethylase; hTNFα – human tumor necrosis factor α; ICAM-1 – intercellular adhesion molecule-1; IL-8 – interleukin 8; TNF-α – tumor necrosis factor aα.


### 2.2. Cytotoxicity of Anatoxins (Table2)

Anatoxins-a are two types of low molecular bicyclic amino alkaloids: anatoxin-a (ANTX) and homoanatoxin-a (hANTX). The 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].Table 2
Cytotoxicity of ANTX, hANTX and ANTX(S).
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.RAW 264.7, BV-2, N2aMTT assay, caspase-glo 3/7 assay, ELISA, TNF-α measurementMC-LR, CYN, ANTX-aMurine macrophage-like RAW264.7, immortalised microglial BV-2, neuroblastoma N2a cell linesCYN, MC-LR and ANTX in a mixture are 3–15 times more potent at inducing apoptosis and inflammationTNF-α[15]Oocytes, M10 cellsPatch-clamp,86Rb + influxANTXXenopus oocytes, human hepatoma cell lineα7-nAChR agonist with EC50 = 0.58 μM (nicotinic current in oocytes), α4β2-nAChR EC50 : 48 nM by 86Rb + influx in M10 cellsα7-nAChR, α4β2-nAChR, Ach[16, 17]GH4C145Ca2+ influx, [3H]-ACh release,hANTX water extract, 1–20 mg/mLRat anterior pituitary cell linehANTX-activated voltage-gated Ca2+ channels and AChR releaseVoltage-gated Ca2+ channels, AChR[18]Chromaffin cell cultureHPLCANTX 0.1–100μMBovine adrenal chromaffin cell cultureCatecholamine release activation above 0.3μM ANTXSecretion of catecholamines[19]Abbreviations: Ach – acetylcholine; AChR – acetylcholine receptor; CYN – cylindrospermopsin; HPLC – high-performance liquid chromatography; MC-LR – microcystin-LR; MTT – 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; nAChR – nicotinic acetylcholine receptor.


### 2.3. Cytotoxicity of BMAA (Table3)

β-N-methylamino-L-alanine (BMAA) is an environmental nonprotein and toxic amino acid that may harm nervous system via oxidative stress, binding to neuromelanin, forming high toxic metabolites like formaldehyde or inhibiting enzyme activity of glutathione reductase, β-amilase, catalase, and RNase H, and in this way to provoke sporadic neurodegenerative development, such as Alzheimer's disease and amyotrophic lateral sclerosis [20, 27, 28]. In addition, BMAA generates a carbamate, which is neurotoxin because it acts as ionotropic and metabotropic glutamate receptors agonist [21] and references therein.Table 3
Cytotoxicity of BMAA.
Cell typeAssayConditionsTissue of originMain effectsRef.HepG2 cells, Caco-2Isotopically labelled amino acids; metabolic activity; apoptotic and necrotic assaysHuman hepatocellular carcinoma and human colorectal epithelial adenocarcinoma cell lineBMAA did not affect the common proteinogenic amino acid metabolic pathways; in the presence of amino acids cellular uptake of BMAA is substantially reduced[20]SH-SY5YLDH assay; qPCR; Western BlotL-BMAA 1 mM/17 hHuman neuroblastoma cellsConversion of procaspase-3 (32 kDa) to active caspase-3 p17 and apoptosis[21]SH-SY5YLDH assay; qPCR; Western BlotL-BMAA 1 mM/17 h and longer for 24–96 hHuman neuroblastoma cellsMisincorporation of L-BMAA protein aggregation, upregulation of lysosomal enzymes and apoptosis; proteolitic stress in prolonged exposure[22]SH-SY5YLow L-BMAA (≥0.1 mM)/48 h; high L-BMAA (≥2 mM)/48 hHuman neuroblastoma cellsLow L-BMAA increases protein ubiquitination, 20S proteasomal and caspase 12 activity, stress marker CHOP expression; enhances phosphorylation of elf2α in SH-SY5Y cells; high L-BMAA increases ROS and protein oxidization[23]OECLDH assay, MTS assay, Ca2+ influx assay, DCFDA assay for ROS, DNA damage assayBMAA 0.1–3 mM/48 hRat olfactory ensheathing cells (special glial cells)Cytotoxic, increases Ca2+ influx, and ROS production; disrupts mitochondrial activity[24]Primary neuronsLDH assay, MTS assay, Ca2+ influx assay, DCFDA assay for ROS, DNA damage assayBMAA 0.1–1 mM/48 hPrimary neurons were obtained from 16 to 19 old foetuses and mixed brain cell culturesBMAA increases Ca2+ influx and DNA damage, enhances production of ROS, disrupts activity of mitochondria[25]SH-SY5Y, HT22, Neuro-2aMTT assay, siRNA transfection, flow cytometry for DNA contentBMAA 1–3 mM/12, 24, and 48 hHuman neuroblastoma cells; mouse hippocampal cell line; mouse neuroblastoma cell lineL-BMAA-induced ER-stress mediated apoptosis via upregulation of ER-stress sentinels, phosphorylation of JNK, p38 and ERK, CHOP activation[26]SH-SY5Y, MRC-5, HUVECLiquid chromatography tandem mass spectrometry, radiolabeled3H-BMAA assay, LDH assay,0.3 mM BMAA and 300 mM L-serine for 96 hoursHuman neuroblastoma and human lung fibroblast cell line, human umbilical endothelial cellsBMAA is misincorporated in place of L-serine into human proteins and this is inhibited by L-serine[22]Abbreviations: CHOP – C/EBP homologous protein; DCFDA – 2′,7′-Dichlorofluorescin diacetate assay; ER – endoplasmic reticulum; JNK – c-JunN-terminal kinase; MTS – 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.


### 2.4. Cytotoxicity of CYN (Table4)

CYN is a cyclic quinidine alkaloid combined with hydroxymethyl uracil [49]. It has two epimers, which are equally toxic and are differentiated by the hydroxyl bridge CYN and 7-epi-CYN, and an additional variant 7-deoxy-CYN occurs in natural waters [49]. CYN has been classified mainly as hepatotoxin, but it has also neurotoxic and genotoxic effects and inhibits protein synthesis [3]. It targets kidneys, lungs, heart, spleen, eyes, ovaries, T-cells, neutrophils, and vascular endothelium [50]. CYN may induce oxidative stress, decrease cell viability, and damage mitochondria (discussed by Chichova et al. [35]).Table 4
Cytotoxicity of CYN.
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.CaCo-2Neutral red uptake1.1 mg/g dw; 0.08–1.25 mg dw/mL/48 hImmortalized human colorectal adenocarcinoma cell lineCytotoxicity, EC50 : 0.4 ± 0.1 mg dw/mL[29]CaCo-2Transepithelial electrical resistance (TEER)CYN 1–10 µM/3–24 hImmortalized human colorectal adenocarcinoma cell line16.7–20.5% intestinal permeability in 24 h; epithelial integrity not significantly altered[30]CaCo-2Permeability of pseudoepithelial layerCYN 1.9–48 µM/24–48 hImmortalized human colorectal adenocarcinoma cell lineApparent permeability: 3.45 × 10−7 cm/s (absorptive), 6.41 × 10−7 cm/s (secretive); epithelial permeability (increase): Tenfold (absorptive), 0.7-fold (secretive);[31]CaCo-2, NCI-87, HCT-8, HuTu-80, Vero, C3A, HepG2MTT assay for cell viabilityCYN 0.25–5 µM/1–7 daysGastro-intestinal and hepatic cell linesCYN sensitivity decreased in cell lines as follows: Gastric > duodenal > ileal > colonic; EC50 is 6.5 ± 3.3 µM for CaCo-2[32]CaCo-2, HepaRGCytokinesis-block micronucleus assayCYNThe same human hepatocyte cell lineCYN increased the frequency of micronuclei in binucleated cellsCytochrome P450[33]CaCo-2Bradford assay for total protein content, MTS reduction for cell viability, GSH and ROS content, electron microscopyCYN 0.7–96 µM/24–48 hThe sameLipid degeneration, mitochondrial damage, nucleolar segregation with altered nuclei, ultrastructurMembranes, mitochondria, nuclei, endosomes[34]HIEC-6MTT assayCYN 1.0–11 µM/24 hHuman intestinal epithelial cell lineReduced cell viability by 13.4% and 21.8%[35]mESReal-time PCR (RT-PCR)CYN 0–1μg/mL/24–168 hUndifferentiated mouse embryonic stem cellEC50 0.86 μg/mL/24 h, LOEC is 1 μg/mLOct4 Brachyury NestinReference [36]HepG2MTS test, flow cytometry, RT-PCR0.125, 0.25, 0.5 µg/mL CYN + MC-LR, 1 µg/mL/24 and 72 hHuman hepatocellular carcinoma cell lineDNA double-strand breaks after 72 h, upregulation of CYP1A1 by CYN and CYN + MC-LR via CDKN1A and GADD45 A genes, cells arrested in G0G0/G1 phaseDNA[37]Rat hepatocytesLDH leakage; cysteine, ATP, and GSH assayCYN 2.5–5 µM/12 hRat hepatocyte cell cultureInhibition of GSH synthesisGSH, cytochrome P450[38]Mouse hepatocytesLDH leakage, protein synthesisCYN 2.5–5 µM/4–18 hMouse hepatocyte cell cultureInhibition of LDH leakage, max at 0.5 µM CYN; CYN, 1–5 µM lead to 52%–82% cell deathProtein synthesis, cytochrome P450[39]HepG2MTS assay, live/dead staining, qPCR, flow cytometry, confocal z-stack imagingCYN 0.125, 0.25, 0.5μg/mL/72 hHuman hepatocellular carcinoma cell lineCYN deregulated genes for phase I and II enzymes, for cell proliferation; apoptosis and DNA damage responseDNA, expression of many enzymes[40]WIL2-NSCentromere staining, PCR, cytokinesis-block micronucleus assayCYN 1, 3, 6, 10μg/mL/24 hLymphoblastoid cell-lineCytogenetic damage by DNA- and kinetochore/spindle-dependent mechanismsCentromere, micronuclei[41]HepG2LDH leakage, MTT assay, flow cytometry, immunocytochemical stainingCYN 0.1–0.5 µg/mL/24–96 hHuman hepatoma cellsGenotoxic effect by DNA double-strand breaksDNA[42]CLCCYN 0.1, 0.5, 1 µg/mL/24 hCommon carp (Cyprinus carpio L.) leucocyte cell lineDecreased cell membrane integrity, GSH/GSSG ratio, inhibited cell proliferation, DNA damage, increased ROS and ATP levels (1 µg/mL)Micronuclei, GSH, ATP, SOD[43]HepG2MTS assay, qPCR, flow cytometryCYN 0.5μg/mL/24 or 72 h, biphenols 10 μg/mLHuman hepatoma cellsDeregulation of some genes was more pronounced after exposure to the mixtureDNA[44]CaCo-2Immortalized human colorectal adenocarcinoma cell lineApparent permeability of the pseudoepithelial cell layer to MC-LR[45]A7r5AO/EB staining assay and comet assay, flow cytometry, qRT-PCRCYN 20, 200, 2000 nM/24 hRat vascular smooth muscle cellsCYN induced apoptosis in a dose-dependent manner, DNA damageActin, p53, Bax/Bcl2, SOD, CAT and GPX[46]LLC-PK1Flow cytometry, qRT-PCR1.0μg/mLRenal epithelial cells derived from proximal tubulesCYN induced necrosis and increased gene expression of Na+/K + –AtpaseNa+/K+-ATPase activity[47]Human keratinocytesLDH leakage, WST-1 cell proliferation assay, Scratch test, crystal violet assay1, 10μg/mL for 24/48hPrimary human keratinocytesCYN induced cytotoxicity, impaired migration, and inhibition of proliferation[48]Abbreviations: AO/EB staining – acridine orange/ethidium bromide staining; ATP – adenosine triphosphate; CAT – catalase; GPX – glutathione peroxidase; GSH – glutathione; MTS – 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; RP-PCR–reverse transcription polymerase chain reaction; RT-qPCR – quantitative reverse transcription polymerase chain reaction; SOD – superoxide dismutase.


### 2.5. Cytotoxicity of Depsipeptides (Table5)

Depsipeptides are palmyramide A (Pal A), apratoxinD (AT D), coibamide A (CoA), ichthyopeptins A (Ich A) and B (Ich B), kahalalide F (KF), 4-Fluoro-3-methyl-benzylamino-KF (KF2), morpholin-4-yl-benzylamino-KF (KF4), homodolastatin 16 (HD16), lagunamide C–Lag C, pitipeptolides–Pit A-F, aurilides and wewakpeptins A-D. Depsipeptides show cytotoxic activity and are protease inhibitors selective for chymotrypsin, leukocyte, and pancreatic elastases. They negatively influence the metabolism of human astrocytes [63].Table 5
Cytotoxicity of depsipeptides.
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.N2a, NCI H-460MTT assayPal A IC50: 17.2 μM/24 h; 39.7 μM/48 hNeuro2a mouse neuroblastoma cells; human lung carcinoma cellsBlockage of the voltage-gated sodium channel, modest cytotoxic effects.Voltage-gated sodium channel[51]NCI H-460MTT reduction20μg/wellHuman lung carcinoma cellsCytotoxicity, IC50: 2.6 nM/48 hG1-phase cell cycle arrest, apoptosis[52]60 cancer cell linesFlow cytometryHuman cells from lung, colon, leukemia, melanoma, CNS, ovarian, prostate, breast and renal cancersCytostatic and cytotoxic effects – increase the number of cells in G1, little change in G2/M and loss of cells in S-phase. GI50 for CoA: 2.8 nM to MDA-MB-231 7.4 nM to LOX IMVI 7.4 nM to HL-60(TB)Novel unknown mechanism; no effect on tubulin or actin in cytoskeletal assays[53]MDCK cells infected with influenza virus A/WSN/33/London (H1N1)Dye uptake assay using neutral redIch A and B in nontoxic conc. 12.5–100μg/mL/30 min.Canine kidneyAntiviral activity, IC50: 12.5μg/mLNon-trypsin protease inhibition[54]60 human cancer cell lines (NCI-60 cell lines)Biokinetics reader, fluorescence detection, acute toxicity determination, MTT assay, hollow fiber assayHuman leukemia, melanoma, lung, colon, CNS, ovarian, prostate, breast and renal cancer cell linesAntitumor and antifungal activities; GI50 for NCI-H322M: KF2–0.131 μM; KF4–0.133 μM; KF–0.191 μM; GI50 = 0.123 μM for human prostate (DU-145); GI50 = 0.453 μM for breast cancer (HS 578T) cell lines. IC50 for C. neoformans: KF–1.53 μM, KF2–0.95 μM—[55]WHCO1, WHCO6, ME180MTT assayWHCO1,06–esophageal and ME180–cervical cancer cellsCytotoxicity: IC50 for HD16 WHCO1–4.3 μg/mL; WHCO6–10.1 μg/mL; ME180–8.3 μg/mL—[56]P388, A549, PC3, HCT8, SK-OVMTT assay, scintillation countingP388-murine leukemia, A549-lung carcinoma, PC3-prostate cancer, HCT8 -ileocecal colorectal adenocarcinoma and SK-OV-ovarian cancer cellsCytotoxicity and antimalarial activity; IC50 for cancer lines: P388–24.4 nM; A549–2.4 nM; PC3–2.6 nM; HCT8–2.1 nM; SK-OV–4.5 nM; IC50 for Plas. Falciparum–0.29 μMMitochondria-induced apoptosis, lag C selectively binding to the prohibitin[57]HT-29, MCF7MTT assay, disc diffusion assayHT-29 colon adenocarcinoma, MCF7 breast cancer cellsCytotoxicity and antimycobacterial activity againstM. tuberculosis. For HT-29 IC50: Pit A–13 μM; Pit B–13 μM; Pit C–67 μM; Pit D – >100 μM; Pit E–75 μM; Pit F–87 μM and for MCF7 IC50 : Pit A–13 μM; Pit B–11 μM; Pit C–73 μM; Pit D – >100 μM; Pit E – >100 μM; Pit F–83 μM-[58]HeLa cellsWST-1 assay, Immuno-precipitation100 nM aurilideHuman cervical cancer cellsCytotoxicity, mitochondria-induced apoptosisProhibitin 1, optic atrophy 1[59]NCI–H460, neuro-2aMTT reduction.NCI–H460 – human lung tumor, neuro-2a – mouse neuroblastoma cell linesCytotoxicity for NCI–H460 LC50 is: Wew A–0.65 μM; wew B–0.43 μM; wew C–5.9 μM; wew D–3.5 μM; for neuro-2a LC50: Wew A – 0.49 μM; wew B–0.20 μM; wew C–10.7 μM; wew D–1.9 μM[60]60 human cancer cell lines (NCI-60 cell lines), MEFsImmunoblot, MTT assay, Trypan blue exclusion, LDH assay, caspase activity assay, autophagy assays, EGF receptor degradation assaysHuman cancer cells from leukemia, melanoma, lung, colon, CNS, ovarian, prostate, breast, renal cancersCytotoxicity, apoptosis, and inhibition of cell growth. EC50 cytotoxicity is < 100 nM for human U87-MG and SF-295 cells, and for mouse embryonic fibroblastsCaspase-3, extensive cytoplasmic vacuolization, mTor-independent pathway[61]HCC2218, UACC-893, T-47D and >50 othersGrowth inhibition assay, immune-precipitation study, SEAP secretion assayHuman breast, ovarian, endometrial, pancreatic, skin, lung, and colon cancer cell lines; rat pancreatic exocrine cell lineCytotoxicity, blocking of cotranslational translocation. IC50 = 5–50 nM for different cell typesSec61 in the ER membrane.[62]Abbreviations: EGF – epidermal growth factor; SEAP – secreted embryonic alkaline phosphatase.


### 2.6. Cytotoxicity of Lipopolysaccharides (LPS, Table6)

LPS consist of lipid A, the core polysaccharides (mainly glucosamine) and an outer polysaccharide chain, and are common compounds of the cell walls of cyanoprokaryotes and Gram-negative bacteria [49]. They have an inflammatory effect and promote cytokine secretion [3].Table 6
Cytotoxicity of LPS.
Cell typeAssayConditionsTissue of originMain effectsRef.MicrogliaSuperoxide anion (O2−) generation, cell viability by LDH release, thromboxane B2 (TXB2), immunoassay, gelatinase zymography for matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-9 (MMP-9), rat-specific ELISA for cytokines and chemokinesMicrocystis aeruginosa LPS strain UTCC 299; 0.1–100,000 ng/mL/17 h E. coli LPS (0.1–100 ng/mL) as controlRat neonatal brain microgliaEnhanced O2− generation, limited inflammatory mediator generation; MMP-9, macrophage inflammatory protein-2 (MIP-2/CXCL2) release, TXB2, concurrent with maximal O2− generation; elevated TXB2, MMP-9, tumor necrosis factor α (TNF-α), interleukin 1-α (IL-1α), and interleukin-6 (IL-6), macrophage inflammatory protein 1α (MIP-1α/CCL3), and MIP-2/CXCL2; LPS activates brain microglia in vitro and the release of O2−, inflammatory mediators[64]Microglia0.1–100 000 ng/mL Oscillatoria sp. LPS; 17 hRat neonatal microgliaClassical and alternative activation; pro-inflammatory and anti-inflammatory mediator release[65]MicrogliaScytonema javanicum and S. ocellatum LPSRat neonatal microgliaConcentration-dependent O₂-, MMP-9, IL-6 TNF-α, MIP-2/CXCL-2, CINC-1/CXCL-1, MIP-1α/CCL3, IL-10 release[66]Meningioma cells and meningioma–primary human macrophageSandwich immunoassayCyanobacterial LPS antagonist (CyP) 1–20μg/monolayerHuman meningioma cells and meningioma–primary human macrophage co-culturesCyanobacterial LPS inhibits cytokine production and augments the anti-inflammatory response when combined with benzylpenicillin[67]MicrogliaImmunocytochemical and immunofluorescent assay, ELISA, immunoblotting, live-cell imaging analysesCyanobacteria-derived TLR4 antagonist—a highly (95%) purified form of LPS-like molecule from Oscillatoria planktothrix sp. 20μg/mL for 24 h,Primary cultures from mouse spinal cordsTLR4 antagonists could be considered as a candidate of protective agents for motor neurons in degenerative diseases[68]Spleen cellsHot-water extract ofSpirulina platensisIn vitro cultures of murine spleen and thymus cellsIncreased proliferation of spleen cells; enhanced IL-1 production from peritoneal macrophages[69]hTHP-1ELISA, real-time PCRCyanobacterial LPS antagonist (CyP) fromOscillatoria planktothrix FP1; 10 µg/mL/5 hHuman THP-1 monocytic cell lineCyP is able to induce cross-tolerance toE. coli LPS by inhibiting TNF-α production[70]


### 2.7. Cytotoxicity of Lyngbyatoxins (Table7)

Lyngbyatoxins were first identified from Moorea producens (formerly Lyngbya majuscula). They are tumor-promoting agents which bound eucaryotic protein kinase C (PKC) isozymes [3].Table 7
Cytotoxicity of lyngbyatoxins.
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.FibroblastsMTT assay, [3H]-thymidine incorporation assay15 mg/mL (w/v) of the cyanobacterial extract/4 h or 24h for MTT test; 24 h for [3H]-thymidine incorporation assayPrimary mouse thymus fibroblasts80% inhibition of cell proliferation, morphology and attachment in 24 hDNA, cell membrane, cytoskeleton[70]FLNormal amniotic cells, humanStimulation of MTT reduction after 4h > 40% vs control cells; decreased cell viability to 32% of controls in 24 hA2058Human metastatic melanomaCytotoxic in 24 hRDHuman embryonic myosarcomaCytotoxic in 24 h3T3Mouse embryonic fibroblasts92% inhibition of cell proliferationL1210MTTLyngbyatoxin A and 12-epi-lyngbyatoxin A/18 hMouse lymphocytic leukemia cell lineCytotoxic effect; IC50 = 8.1 μM lyngbyatoxin A; IC50 = 20.4 μM 12-epi-lyngbyatoxin APKC isozymes[71]HL-60 CTest of induction of cell adhesionLyngbyatoxin A and debromoaplysia toxin/48 hHuman promyelocytic leukemia cellsFor 50% cell adhesion to the flasks—7 ng/mLLyngbyatoxin A and 700 ng/mL debromoaplysiatoxinCell membrane[72]DS 19Test of inhibition of terminal differentiationMouse erythroleukemia cells transformed by Friend leukemia virus strain 745AInhibition of terminal differentiation in 50% of the cells with 0.35 ng/mLLyngbyatoxin A and 150 ng/mL debromoaplysiatoxinNeuro-2aMTT24 hMouse neuroblastoma cellsCytotoxicity IC50 = 2.2 μM of hermitamides A; IC50 = 5.5 μM hermitamides B[73]CHOPatch-clamp0.1–30μM neo-debromoaplysia toxin G and HChinese hamster ovary cellsPotassium channel Kv1.5 block; IC50 = 1.79 μM debromo aplysiatoxin G and IC50 = 1.46 μM debromoaplysia toxin HVoltage-gated potassium channels Kv1.5 (KCNA5)[74]


### 2.8. Cytotoxicity of MCs (Table8)

MC are cyclic nonribosomal heptapeptides with low molecular weight (800–1100 Da), which contain several uncommon nonproteinogenic amino acids such as N-methyldehydroalanin (MDHA) derivatives and the uncommonβ-amino acid 3-amino-9-methoxy-2,6,8-trimethyldeca-4,6-dienoic acid (ADDA). MC are lipophilic toxins very resistant to hydrolysis, oxidation, and high temperatures. The main route of human exposure is the ingestion of contaminated drinking water, consumption of contaminated food or algal dietary supplements, and body contact, while more occasional routes are hemodialysis and inhalation. MC are classified mainly as hepatotoxins because they block eucaryotic PP (PP1, 2A and phosphoprotein phosphatases PPP4, PPP5) [2] through irreversible covalent binding [97]. Chronic and subchronic exposure to MC seems to be tumor promoting because they can increase the incidence of hepatic tumors in humans. MC could also enhance the oxidative stress. Additional target of MC in high concentrations is the ß-subunit of ATP synthase, causing mitochondrial apoptotic signaling. MC have hepatotoxic and tumor promoting action [3].Table 8
Cytotoxicity of MC.
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.CaCo-2Immuno-localization of MC uptakeMC-LR 1–75 µM/30 min–24 hImmortalized human colorectal adenocarcinoma cell lineArtificial epithelial cell layer is highly permeable to MC-LR[45]CaCo-2Gene expression, transcriptomicsMC-LR 10–100 µM/4–24 hThe sameOxidative stressERK/MAPK and cell cycle pathway molecules[75]CaCo-2Comet assay, MTT assay (for viability)MC-LR 0.2–10 µM/4–48 hThe same20% damaged DNA after 0.2 µM/4 h MC-LR; 40% reduced cell viability after MC-LR 10 µM/48 h,DNA[76]CaCo-2Protein phosphatase (PP) inhibition, LDH leakage, cell morphology and proliferation1–50 µM MC-LR, -LF and -LW for 22–48 hThe samePP inhibition—3.0 nM MC-LF, 3.8 nM MC-LW, 1.0 nM MC-LR, EC50 of LDH leakage: 25% (50 µM MC-LR), 36% (MC-LW), 51% (MC-LF), chromatin cell shrinkage, condensation, membrane blebbing, and cytoskeletal reorganizationPP, cell membrane, chromatin, cytoskeleton[77]CaCo-2Bradford assay, MTS reduction (for viability), neutral red uptakeMC-LR, –RR and -YR, 50–200 µM/24–48 hThe sameEC50 reduction of total protein content by MC-LR 111.1 ± 3 µM/24 h and MC-RR ˃200 µM/48 h; neutral red uptake—MC-YR 57.3 µM/48hProtein synthesis[78, 79]CaCo-2Immuno-localization of microcystinsMC-LR, –RR, 1–50 µM/30 min–24 hThe sameFacilitated MC uptake in <1 h by organic anion transporters, active excretionOrganic anion transporters 3A1 and 4A1[80]HIEC-6Cell counting Kit-8 for viability, western blot, TEER, PP2A activityMC-LR 0–50 µM/6–24 hHuman intestinal (colon) epithelial cell lineViability–12.5 µM/24 h; TEER at 50 µM/12 h and at 12.5 µM/24 h; apoptosis at 12.5 µM/24 h; western blot at 12.5 µM/24 h; occludin; claudin not affected), 25 µM/24 h; ZO-1; PP2A activity decreases from 12.5 µM/24 hPP2A, occludin, claudin[81]HEK293Western blot, luciferase assay, rtPCRMC-LR 10 µM/24 hHuman embryonic kidney cellsPP2A inhibition, enhanced proto-oncogene C-myc expressionPP2A, c-Myc protein, proto-oncogene C-myc[82]NCCPP2A, PP2B, PP2C activity, western blot, Akt, p38, JNK, PI3K assays, genechip analyses;MC-LR, 0.0001–1.0 µg/24 hImmortalized colorectal crypt cellsConstitute activation of Akt/p38 and JNK/MAPK pathwaysAkt, p38, JNK[83]HBE1, 16HBE14o-RT-PCR, western blot, RTCA, neutral red uptakeMC-LR 1–20 µM/48 hHuman bronchial epithelial cell linesNo effect on viability, ERK1/2 and p38 activities were not changedERK1/2 and p38 not influenced[9]DLD-1, HT-29Western blot, RT-qPCR, knockdown of SMAD2 by siRNA, migration and invasion assayMC-LR, 0.1–50 nM/24 hHuman colorectal cancer cellsInduction of SMAD2 signal transducer and transcriptional modulating protein expression, its activating phosphorylation by PI3K/Akt, increased migration (epithelial-mesenchymal transition of both cell types)PI3K/Akt, SMAD2,[84]BALB/cmRNAMC-LR 1–1000 nmol/L/6 hMouse peritoneal macrophagesDecreased transcription of mRNA for iNOS, IL‐1β, TNF‐α, GM‐CSF, and IFN‐γ; reduced inflammatory response to LPSiNOS, IL‐1β, TNF‐α, GM‐CSF and IFN‐γ[85]RAW 264.7 macrophagesWestern blot, ELISAMC-LR, 1–1000 nmol/L/30 min–24 hAbelson leukemia virus-transformed cell line from BALB/c miceActivation of NF-κB with 1000 nM and ERK1/2 with 100 nM; TNF-α synthesis (1 nM)NF-κB, ERK1/2, TNF-α[86]HepG2RT-qPCR, Western blot, MTT assay, mitochondrial membrane potential (MMP)MC-LR, 0.01–5 µM/3, 6, 12 and 24 hHuman hepatocellular carcinoma cell lineMMP loss, SOD induction in hypoxia, inhibitory apoptosis protein (c-IAP2) up-regulated in normoxic conditionMitochondrial dehydrogenase, SOD, c-IAP2[87]A549MTT assay, PP2A activity, Western blot, proliferationMC-LR, 0.5–10 µM/24 hHuman non-small-cells lung cancer cellsRearrangements of filamentous actin and microtubules due to PP2A/C (>1 µM) and p38 MAPK activation (0.5–10 µM); p-Blc-2, p-Bad (1.0–10 µM)Microtubules and filamentous actin (cytoskeleton), PP2A/C, p38,[88]HEK293Western blot, cell detachment, PP2A activity, MTT assayMC-LR, 0.5–10 µM/24 hHuman embryonic kidney cellsPP2A inhibition (>5 µM); PP2A activation (1–2 µM); cell anoikisPP2A catalytical and regulatory subunits[89]PC12Western blot, PP2A activity, immuno-fluorescenceMC-LR, 0.1–10 µM/6 hPheochromocytoma cells of the rat adrenal medullaRearrangement of filamentous actin and microtubules due to PP2A (>0.5 µM) and p38 MAPKPP2A, p38 MAPK, HSP27[90]HL7702PP2A activity, western blot, immuno-fluorescenceMC-LR, 5 or 10 µM for 30 min to 24 h;Human normal liver cell lineActivation of p38 MAPK, JNK and ERK1/2, HSP27-sensiitive cytoskeleton reassembly, PP2A inhibition in 6–24 h; activated phosphorylation of tau (by P38 MAPK) and VASPp38 MAPK, JNK, ERK1/2, PP2A; tau and VASP components of cytoskeleton[91, 92]SMMC-7721PP2A activity, western blot, PKA activity and Rac1/Cdc42 activity immuno-fluorescence, immuno-precipitationMC-LR, 0.5–10 µM/24 hHuman liver cancer cell linep-HSP27, p-VASP and p-cofilin contributed to cytoskeleton change; PP2A inhibition (>0.5 µM); disorder of cytoskeletonHSP27, VASP, cofilin, PKA, Rac1, PP2A[93]HepaRGCytopathic effects, RNA quantified by Agilent RNA 6000 Nano kitMC-LR, –RR 10, 100 and 1000 ng/2 hHuman hepatocyte cell lineIncrease of RNA of apoptotic and inflammatory gene; many cellular pathways activated[94]HL7702Real-time cell analyzer (RTCA) proliferation, cell cycle analysis, western blot, PP2A activity, MTT assay, immuno-fluorescenceMC-LR, 1, 5, 10 µM/1–96 hHuman normal liver cell lineMC-LR promoted HL7702 cell proliferation (36–48 h); activation of Akt/S6K1 cascade; PP2A activity (>1 µM), hyper-phosphorylation of Bcl-2, Bad, c-Myc and c-Jun, 1–10 µMPI3K/Akt/S6K1, hyper-phosphorylation of Bcl-2, Bad, c-Myc and c-Jun[95]HBEMTT and Annexin V/PI assay, ROS and MMP measurements, western blotMC-LR, 1, 10, 20, 30, 40 µg/mL/24, 48 hHuman bronchial epithelial cellsInducing mitochondria-dependent apoptosis (1–40 µg/mL), MMP decreases at 10 µg/mLCaspases[89]Huh7MC-LR, 0.5–50μM/6–72 hHuman hepatoma cells5μM MC-LR induced PP2A mRNA expression, p-CREB, expression of NF-κB, IFN-α, and several INFα-stimulated genes are activatedNF-κB, p-CREB, DNA[96]Abbreviations: CREB – cAMP responsive element-binding protein; ERK/MAPK – extracellular signal-regulated kinase/mitogen-activated protein kinase; GM‐CSF – granulocyte macrophage colony-stimulating factor; IFN-γ – interferon gamma; iNOS – inducible nitric oxide synthase; JNK–c – Jun N-terminal kinases; mRNA – messenger RNA; siRNA – small interfering RNA, VASP – vasodilator-stimulated phosphoprotein.


### 2.9. Cytotoxicity of Nodularins (Table9)

Nodularins (NODs) are cyclic nonribosomal pentapeptides and contain several unusual nonproteinogenic amino acids such as N-methyl-didehydroaminobutyric acid and theßβ-amino acid (all-S, all-E)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (ADDA). Ten variants have been discovered with nodularin-R being the predominant toxin variant. NODs are relatively stable compounds, with low sensitivity to light or temperature. NOD affects hepatocytes binding their PPs by noncovalent bonds, which increases the rate of phosphorylation. They are often attributed to gastroenteritis, allergic irritation reactions, and liver diseases. Nodularin-R is the most notorious as a potent hepatotoxin that may cause serious damage to the liver of humans and other animals. NODs have similar effects as microcystins and weak carcinogenicity [3].Table 9
Cytotoxicity of nodularins.
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.CLCFluorometric cell membrane integrity, cell viability and ROS measurements, caspase-glo 3/7 assay, ELISANOD, 0.001, 0.01, 0.05, 0.1μg/mL/24 hCarp leukocyte cell line and head kidney leukocytesCell viability, membrane integrity at 0.1μg/mL, DNA fragmentation and caspases 3/7 activation at > 0.1 μg/mL, ROS increase in 60 min in >0.01 μg/mL, GSH decrease at >0.001/24 hGSH/GSSG, DNA, membranes, caspases[98]CLC and kidney leucocytesFluorometric cell viability, ROS and nitrogen species (NS) measurements0.001, 0.01, 0.05, or 0.1μg/mL/24 hCarp leukocyte cell line, kidney leukocytesCytotoxicity ≥0.05μg/mL, ROS and NS increase, expression of TNF-α, IL-10, less TGF-βDNA expression[99]HepG2Micronucleus assay, Flow cytometry, comet assay, DNA damageNOD, 1–10μg/mL, for 6, 12, 24, 48 hHuman hepatoma cell lineDNA damage >1μg/mL, apoptosis from1 μg/mL/48 hDNA, cellular and mitochondrial membranes[100]HepG2RT-PCR, siRNA, flow cytometry, transfection of NF-κB immunoblotingNOD, 2.5, 5, 7.5, 10μM/24 hHuman hepatoma cell lineInduces fas receptor (fas) and fas ligand (FasL) expression and apoptosisNF-κB pathway, fas, FasL[101]HepG2 and Huh7ATF-6 activity qPCR, TNF-α ELISA, immunoblottingNOD, 0.1, 1, 5μM for 24, 48 and 72 hHuman hepatoma cell linesInduction of TNF-α protein, CAAT/enhancer-binding protein-homologous,TNF-α, ERK 1/2 MAPK, ER, IL-8, CHOP ER-stress—2.5 nM[102]HepG2qPCR, MTT assay, comet assay, cytokinesis micronucleus assayNOD, 0.01, 0.1 and 1μg/mLHuman hepatoma cell lineDNA damage; apoptosis (BAX, BCL2) genes, ROS increase, oxidative stressDNA, ROS[103]


### 2.10. Cytotoxicity of Retinoids from Cyanobacteria (Table10)

Retinol, a novel retinoic acid (RA) analogue 7-hydroxy RA, 4-oxo-RA, and several analogues were identified in cyanobacterial blooms [110]. They act as RA receptors that may cause different malformations, as well as to have a teratogenic effect on aqueous animals.Table 10
Cytotoxicity of cyanobacterial retinoids.
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.P19/A15Bioluminescence reporter assay; calcein AM cell viability assay0.25, 0.5, 1 and 2 g dm/l/24 h; water extracts 2.5x–20x environmental water/24hMurine embryonal carcinoma cells stably transfected with firefly luciferase geneRetinoid-like activity, max 263 ng retinoid eq/L; cytotoxic effect at 20xRAR[104]P19/A15Bioluminescence reporter assay;0.25, 0.5, 1 and 2 g dw/l/24hMurine embryonal carcinoma cellsRetinoid-like activityRAR[105]P19/A15RAR/RXR transactivation assayCyanobacterial extracts 0.125–2 g dw/l/24 hMurine embryonal carcinoma cellsRetinoid acid receptor (RAR) activityRAR[106]P19/A15Bioluminescence reporter assayCyanobacterial extracts 0.25–2 g dm/l and exudates 2.5×–20×/24 hMurine embryonal carcinoma cellsRetinoid acid receptor activityRAR[107]P19/A15Bioluminescence reporter assay1x–20x concentrated cyanobacterial and algal exudates/24hMurine embryonal carcinoma cellsRetinoid-like activityRAR[108]HepG2MTT, comet assay, cytokinesis-block micronucleus (cytome) assay0.04–2 mg/mL/24h for MTT, 0.2 mg dm/mL/24h for cytokinesis-block micronucleus assayHuman hepatocellular carcinoma cellsSignificant genotoxic effects of retinoic acid from the extractsDNA[109]


### 2.11. Cytotoxicity of saxitoxins (Table11)

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. They have a neurotoxic effect by blocking voltage-gated sodium channels [3].Table 11
Cytotoxicity of SXT.
Cell typeAssayConditionsTissue of originMain effectsRef.Neuro-2AHPLC, LC-MS/MS, Jellett rapid test, MTT assaySTX, 0.05–200 ng/mLMouse neuroblastoma cell lineScreening assay for determination of toxicity and comparison of various methods for detection of toxins[111]IEC-6, Caco-2HPLC,Gonyautoxin, 100μM for 1–60 minHuman colorectal, adenocarcinoma cell lineIEC-6 cells secrete the toxin, Caco-2 cells absorb it Na+-dependently[112]


## 2.1. Cytotoxicity of Aeruginosins (Table1)

Aeruginosin CT contains as a basic structure 2-carboxy-6-hydroxyoctahydroindol that are serine protease inhibitors [12]. They inhibit trypsin-like serine proteases and for this activity are important in the search for new anticoagulants [13].Table 1
Cytotoxicity of aeruginosins.
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.Huh7 cellsEROD assay, treatment with TNF-αAeruginosin-865A 50 and 100 µmol/LHuman hepatoma cell lineAnti-inflammatory activity by inhibition of IL-8 and TNF-α expression; induce expression of cytochrome P450 1A (CYP1A)DNA[12]HLMVECIL-8 and ICAM-1 assay upon stimulation with human tumor necrosis factorα (hTNF-α)Aeruginosin-865 0.1–100 µg/mL/18 h of 0.1 ngm/L hTNF-α stimulated cellsHuman lung microvascular endothelial cellsAnti-inflammatory activity by down-regulation of IL-8 (EC50 : 4.0 ± 1.7 mM) and intercellular adhesion molecule 1 (ICAM-1; 57.8 ± 15.5 mM)Inhibits NF-kappa B translocation to the nucleus[13]WEHI-13VARLactate dehydrogenase (LDH) cytotoxicity assayAeruginosin-865 10–200 µMMouse fibrosarcoma cellsCytotoxic effect of aeruginosin-865 at 200 µM only[14]Abbreviations: EROD – ethoxyresorufin-O-deethylase; hTNFα – human tumor necrosis factor α; ICAM-1 – intercellular adhesion molecule-1; IL-8 – interleukin 8; TNF-α – tumor necrosis factor aα.


## 2.2. Cytotoxicity of Anatoxins (Table2)

Anatoxins-a are two types of low molecular bicyclic amino alkaloids: anatoxin-a (ANTX) and homoanatoxin-a (hANTX). The 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].Table 2
Cytotoxicity of ANTX, hANTX and ANTX(S).
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.RAW 264.7, BV-2, N2aMTT assay, caspase-glo 3/7 assay, ELISA, TNF-α measurementMC-LR, CYN, ANTX-aMurine macrophage-like RAW264.7, immortalised microglial BV-2, neuroblastoma N2a cell linesCYN, MC-LR and ANTX in a mixture are 3–15 times more potent at inducing apoptosis and inflammationTNF-α[15]Oocytes, M10 cellsPatch-clamp,86Rb + influxANTXXenopus oocytes, human hepatoma cell lineα7-nAChR agonist with EC50 = 0.58 μM (nicotinic current in oocytes), α4β2-nAChR EC50 : 48 nM by 86Rb + influx in M10 cellsα7-nAChR, α4β2-nAChR, Ach[16, 17]GH4C145Ca2+ influx, [3H]-ACh release,hANTX water extract, 1–20 mg/mLRat anterior pituitary cell linehANTX-activated voltage-gated Ca2+ channels and AChR releaseVoltage-gated Ca2+ channels, AChR[18]Chromaffin cell cultureHPLCANTX 0.1–100μMBovine adrenal chromaffin cell cultureCatecholamine release activation above 0.3μM ANTXSecretion of catecholamines[19]Abbreviations: Ach – acetylcholine; AChR – acetylcholine receptor; CYN – cylindrospermopsin; HPLC – high-performance liquid chromatography; MC-LR – microcystin-LR; MTT – 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; nAChR – nicotinic acetylcholine receptor.


## 2.3. Cytotoxicity of BMAA (Table3)

β-N-methylamino-L-alanine (BMAA) is an environmental nonprotein and toxic amino acid that may harm nervous system via oxidative stress, binding to neuromelanin, forming high toxic metabolites like formaldehyde or inhibiting enzyme activity of glutathione reductase, β-amilase, catalase, and RNase H, and in this way to provoke sporadic neurodegenerative development, such as Alzheimer's disease and amyotrophic lateral sclerosis [20, 27, 28]. In addition, BMAA generates a carbamate, which is neurotoxin because it acts as ionotropic and metabotropic glutamate receptors agonist [21] and references therein.Table 3
Cytotoxicity of BMAA.
Cell typeAssayConditionsTissue of originMain effectsRef.HepG2 cells, Caco-2Isotopically labelled amino acids; metabolic activity; apoptotic and necrotic assaysHuman hepatocellular carcinoma and human colorectal epithelial adenocarcinoma cell lineBMAA did not affect the common proteinogenic amino acid metabolic pathways; in the presence of amino acids cellular uptake of BMAA is substantially reduced[20]SH-SY5YLDH assay; qPCR; Western BlotL-BMAA 1 mM/17 hHuman neuroblastoma cellsConversion of procaspase-3 (32 kDa) to active caspase-3 p17 and apoptosis[21]SH-SY5YLDH assay; qPCR; Western BlotL-BMAA 1 mM/17 h and longer for 24–96 hHuman neuroblastoma cellsMisincorporation of L-BMAA protein aggregation, upregulation of lysosomal enzymes and apoptosis; proteolitic stress in prolonged exposure[22]SH-SY5YLow L-BMAA (≥0.1 mM)/48 h; high L-BMAA (≥2 mM)/48 hHuman neuroblastoma cellsLow L-BMAA increases protein ubiquitination, 20S proteasomal and caspase 12 activity, stress marker CHOP expression; enhances phosphorylation of elf2α in SH-SY5Y cells; high L-BMAA increases ROS and protein oxidization[23]OECLDH assay, MTS assay, Ca2+ influx assay, DCFDA assay for ROS, DNA damage assayBMAA 0.1–3 mM/48 hRat olfactory ensheathing cells (special glial cells)Cytotoxic, increases Ca2+ influx, and ROS production; disrupts mitochondrial activity[24]Primary neuronsLDH assay, MTS assay, Ca2+ influx assay, DCFDA assay for ROS, DNA damage assayBMAA 0.1–1 mM/48 hPrimary neurons were obtained from 16 to 19 old foetuses and mixed brain cell culturesBMAA increases Ca2+ influx and DNA damage, enhances production of ROS, disrupts activity of mitochondria[25]SH-SY5Y, HT22, Neuro-2aMTT assay, siRNA transfection, flow cytometry for DNA contentBMAA 1–3 mM/12, 24, and 48 hHuman neuroblastoma cells; mouse hippocampal cell line; mouse neuroblastoma cell lineL-BMAA-induced ER-stress mediated apoptosis via upregulation of ER-stress sentinels, phosphorylation of JNK, p38 and ERK, CHOP activation[26]SH-SY5Y, MRC-5, HUVECLiquid chromatography tandem mass spectrometry, radiolabeled3H-BMAA assay, LDH assay,0.3 mM BMAA and 300 mM L-serine for 96 hoursHuman neuroblastoma and human lung fibroblast cell line, human umbilical endothelial cellsBMAA is misincorporated in place of L-serine into human proteins and this is inhibited by L-serine[22]Abbreviations: CHOP – C/EBP homologous protein; DCFDA – 2′,7′-Dichlorofluorescin diacetate assay; ER – endoplasmic reticulum; JNK – c-JunN-terminal kinase; MTS – 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.


## 2.4. Cytotoxicity of CYN (Table4)

CYN is a cyclic quinidine alkaloid combined with hydroxymethyl uracil [49]. It has two epimers, which are equally toxic and are differentiated by the hydroxyl bridge CYN and 7-epi-CYN, and an additional variant 7-deoxy-CYN occurs in natural waters [49]. CYN has been classified mainly as hepatotoxin, but it has also neurotoxic and genotoxic effects and inhibits protein synthesis [3]. It targets kidneys, lungs, heart, spleen, eyes, ovaries, T-cells, neutrophils, and vascular endothelium [50]. CYN may induce oxidative stress, decrease cell viability, and damage mitochondria (discussed by Chichova et al. [35]).Table 4
Cytotoxicity of CYN.
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.CaCo-2Neutral red uptake1.1 mg/g dw; 0.08–1.25 mg dw/mL/48 hImmortalized human colorectal adenocarcinoma cell lineCytotoxicity, EC50 : 0.4 ± 0.1 mg dw/mL[29]CaCo-2Transepithelial electrical resistance (TEER)CYN 1–10 µM/3–24 hImmortalized human colorectal adenocarcinoma cell line16.7–20.5% intestinal permeability in 24 h; epithelial integrity not significantly altered[30]CaCo-2Permeability of pseudoepithelial layerCYN 1.9–48 µM/24–48 hImmortalized human colorectal adenocarcinoma cell lineApparent permeability: 3.45 × 10−7 cm/s (absorptive), 6.41 × 10−7 cm/s (secretive); epithelial permeability (increase): Tenfold (absorptive), 0.7-fold (secretive);[31]CaCo-2, NCI-87, HCT-8, HuTu-80, Vero, C3A, HepG2MTT assay for cell viabilityCYN 0.25–5 µM/1–7 daysGastro-intestinal and hepatic cell linesCYN sensitivity decreased in cell lines as follows: Gastric > duodenal > ileal > colonic; EC50 is 6.5 ± 3.3 µM for CaCo-2[32]CaCo-2, HepaRGCytokinesis-block micronucleus assayCYNThe same human hepatocyte cell lineCYN increased the frequency of micronuclei in binucleated cellsCytochrome P450[33]CaCo-2Bradford assay for total protein content, MTS reduction for cell viability, GSH and ROS content, electron microscopyCYN 0.7–96 µM/24–48 hThe sameLipid degeneration, mitochondrial damage, nucleolar segregation with altered nuclei, ultrastructurMembranes, mitochondria, nuclei, endosomes[34]HIEC-6MTT assayCYN 1.0–11 µM/24 hHuman intestinal epithelial cell lineReduced cell viability by 13.4% and 21.8%[35]mESReal-time PCR (RT-PCR)CYN 0–1μg/mL/24–168 hUndifferentiated mouse embryonic stem cellEC50 0.86 μg/mL/24 h, LOEC is 1 μg/mLOct4 Brachyury NestinReference [36]HepG2MTS test, flow cytometry, RT-PCR0.125, 0.25, 0.5 µg/mL CYN + MC-LR, 1 µg/mL/24 and 72 hHuman hepatocellular carcinoma cell lineDNA double-strand breaks after 72 h, upregulation of CYP1A1 by CYN and CYN + MC-LR via CDKN1A and GADD45 A genes, cells arrested in G0G0/G1 phaseDNA[37]Rat hepatocytesLDH leakage; cysteine, ATP, and GSH assayCYN 2.5–5 µM/12 hRat hepatocyte cell cultureInhibition of GSH synthesisGSH, cytochrome P450[38]Mouse hepatocytesLDH leakage, protein synthesisCYN 2.5–5 µM/4–18 hMouse hepatocyte cell cultureInhibition of LDH leakage, max at 0.5 µM CYN; CYN, 1–5 µM lead to 52%–82% cell deathProtein synthesis, cytochrome P450[39]HepG2MTS assay, live/dead staining, qPCR, flow cytometry, confocal z-stack imagingCYN 0.125, 0.25, 0.5μg/mL/72 hHuman hepatocellular carcinoma cell lineCYN deregulated genes for phase I and II enzymes, for cell proliferation; apoptosis and DNA damage responseDNA, expression of many enzymes[40]WIL2-NSCentromere staining, PCR, cytokinesis-block micronucleus assayCYN 1, 3, 6, 10μg/mL/24 hLymphoblastoid cell-lineCytogenetic damage by DNA- and kinetochore/spindle-dependent mechanismsCentromere, micronuclei[41]HepG2LDH leakage, MTT assay, flow cytometry, immunocytochemical stainingCYN 0.1–0.5 µg/mL/24–96 hHuman hepatoma cellsGenotoxic effect by DNA double-strand breaksDNA[42]CLCCYN 0.1, 0.5, 1 µg/mL/24 hCommon carp (Cyprinus carpio L.) leucocyte cell lineDecreased cell membrane integrity, GSH/GSSG ratio, inhibited cell proliferation, DNA damage, increased ROS and ATP levels (1 µg/mL)Micronuclei, GSH, ATP, SOD[43]HepG2MTS assay, qPCR, flow cytometryCYN 0.5μg/mL/24 or 72 h, biphenols 10 μg/mLHuman hepatoma cellsDeregulation of some genes was more pronounced after exposure to the mixtureDNA[44]CaCo-2Immortalized human colorectal adenocarcinoma cell lineApparent permeability of the pseudoepithelial cell layer to MC-LR[45]A7r5AO/EB staining assay and comet assay, flow cytometry, qRT-PCRCYN 20, 200, 2000 nM/24 hRat vascular smooth muscle cellsCYN induced apoptosis in a dose-dependent manner, DNA damageActin, p53, Bax/Bcl2, SOD, CAT and GPX[46]LLC-PK1Flow cytometry, qRT-PCR1.0μg/mLRenal epithelial cells derived from proximal tubulesCYN induced necrosis and increased gene expression of Na+/K + –AtpaseNa+/K+-ATPase activity[47]Human keratinocytesLDH leakage, WST-1 cell proliferation assay, Scratch test, crystal violet assay1, 10μg/mL for 24/48hPrimary human keratinocytesCYN induced cytotoxicity, impaired migration, and inhibition of proliferation[48]Abbreviations: AO/EB staining – acridine orange/ethidium bromide staining; ATP – adenosine triphosphate; CAT – catalase; GPX – glutathione peroxidase; GSH – glutathione; MTS – 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; RP-PCR–reverse transcription polymerase chain reaction; RT-qPCR – quantitative reverse transcription polymerase chain reaction; SOD – superoxide dismutase.


## 2.5. Cytotoxicity of Depsipeptides (Table5)

Depsipeptides are palmyramide A (Pal A), apratoxinD (AT D), coibamide A (CoA), ichthyopeptins A (Ich A) and B (Ich B), kahalalide F (KF), 4-Fluoro-3-methyl-benzylamino-KF (KF2), morpholin-4-yl-benzylamino-KF (KF4), homodolastatin 16 (HD16), lagunamide C–Lag C, pitipeptolides–Pit A-F, aurilides and wewakpeptins A-D. Depsipeptides show cytotoxic activity and are protease inhibitors selective for chymotrypsin, leukocyte, and pancreatic elastases. They negatively influence the metabolism of human astrocytes [63].Table 5
Cytotoxicity of depsipeptides.
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.N2a, NCI H-460MTT assayPal A IC50: 17.2 μM/24 h; 39.7 μM/48 hNeuro2a mouse neuroblastoma cells; human lung carcinoma cellsBlockage of the voltage-gated sodium channel, modest cytotoxic effects.Voltage-gated sodium channel[51]NCI H-460MTT reduction20μg/wellHuman lung carcinoma cellsCytotoxicity, IC50: 2.6 nM/48 hG1-phase cell cycle arrest, apoptosis[52]60 cancer cell linesFlow cytometryHuman cells from lung, colon, leukemia, melanoma, CNS, ovarian, prostate, breast and renal cancersCytostatic and cytotoxic effects – increase the number of cells in G1, little change in G2/M and loss of cells in S-phase. GI50 for CoA: 2.8 nM to MDA-MB-231 7.4 nM to LOX IMVI 7.4 nM to HL-60(TB)Novel unknown mechanism; no effect on tubulin or actin in cytoskeletal assays[53]MDCK cells infected with influenza virus A/WSN/33/London (H1N1)Dye uptake assay using neutral redIch A and B in nontoxic conc. 12.5–100μg/mL/30 min.Canine kidneyAntiviral activity, IC50: 12.5μg/mLNon-trypsin protease inhibition[54]60 human cancer cell lines (NCI-60 cell lines)Biokinetics reader, fluorescence detection, acute toxicity determination, MTT assay, hollow fiber assayHuman leukemia, melanoma, lung, colon, CNS, ovarian, prostate, breast and renal cancer cell linesAntitumor and antifungal activities; GI50 for NCI-H322M: KF2–0.131 μM; KF4–0.133 μM; KF–0.191 μM; GI50 = 0.123 μM for human prostate (DU-145); GI50 = 0.453 μM for breast cancer (HS 578T) cell lines. IC50 for C. neoformans: KF–1.53 μM, KF2–0.95 μM—[55]WHCO1, WHCO6, ME180MTT assayWHCO1,06–esophageal and ME180–cervical cancer cellsCytotoxicity: IC50 for HD16 WHCO1–4.3 μg/mL; WHCO6–10.1 μg/mL; ME180–8.3 μg/mL—[56]P388, A549, PC3, HCT8, SK-OVMTT assay, scintillation countingP388-murine leukemia, A549-lung carcinoma, PC3-prostate cancer, HCT8 -ileocecal colorectal adenocarcinoma and SK-OV-ovarian cancer cellsCytotoxicity and antimalarial activity; IC50 for cancer lines: P388–24.4 nM; A549–2.4 nM; PC3–2.6 nM; HCT8–2.1 nM; SK-OV–4.5 nM; IC50 for Plas. Falciparum–0.29 μMMitochondria-induced apoptosis, lag C selectively binding to the prohibitin[57]HT-29, MCF7MTT assay, disc diffusion assayHT-29 colon adenocarcinoma, MCF7 breast cancer cellsCytotoxicity and antimycobacterial activity againstM. tuberculosis. For HT-29 IC50: Pit A–13 μM; Pit B–13 μM; Pit C–67 μM; Pit D – >100 μM; Pit E–75 μM; Pit F–87 μM and for MCF7 IC50 : Pit A–13 μM; Pit B–11 μM; Pit C–73 μM; Pit D – >100 μM; Pit E – >100 μM; Pit F–83 μM-[58]HeLa cellsWST-1 assay, Immuno-precipitation100 nM aurilideHuman cervical cancer cellsCytotoxicity, mitochondria-induced apoptosisProhibitin 1, optic atrophy 1[59]NCI–H460, neuro-2aMTT reduction.NCI–H460 – human lung tumor, neuro-2a – mouse neuroblastoma cell linesCytotoxicity for NCI–H460 LC50 is: Wew A–0.65 μM; wew B–0.43 μM; wew C–5.9 μM; wew D–3.5 μM; for neuro-2a LC50: Wew A – 0.49 μM; wew B–0.20 μM; wew C–10.7 μM; wew D–1.9 μM[60]60 human cancer cell lines (NCI-60 cell lines), MEFsImmunoblot, MTT assay, Trypan blue exclusion, LDH assay, caspase activity assay, autophagy assays, EGF receptor degradation assaysHuman cancer cells from leukemia, melanoma, lung, colon, CNS, ovarian, prostate, breast, renal cancersCytotoxicity, apoptosis, and inhibition of cell growth. EC50 cytotoxicity is < 100 nM for human U87-MG and SF-295 cells, and for mouse embryonic fibroblastsCaspase-3, extensive cytoplasmic vacuolization, mTor-independent pathway[61]HCC2218, UACC-893, T-47D and >50 othersGrowth inhibition assay, immune-precipitation study, SEAP secretion assayHuman breast, ovarian, endometrial, pancreatic, skin, lung, and colon cancer cell lines; rat pancreatic exocrine cell lineCytotoxicity, blocking of cotranslational translocation. IC50 = 5–50 nM for different cell typesSec61 in the ER membrane.[62]Abbreviations: EGF – epidermal growth factor; SEAP – secreted embryonic alkaline phosphatase.


## 2.6. Cytotoxicity of Lipopolysaccharides (LPS, Table6)

LPS consist of lipid A, the core polysaccharides (mainly glucosamine) and an outer polysaccharide chain, and are common compounds of the cell walls of cyanoprokaryotes and Gram-negative bacteria [49]. They have an inflammatory effect and promote cytokine secretion [3].Table 6
Cytotoxicity of LPS.
Cell typeAssayConditionsTissue of originMain effectsRef.MicrogliaSuperoxide anion (O2−) generation, cell viability by LDH release, thromboxane B2 (TXB2), immunoassay, gelatinase zymography for matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-9 (MMP-9), rat-specific ELISA for cytokines and chemokinesMicrocystis aeruginosa LPS strain UTCC 299; 0.1–100,000 ng/mL/17 h E. coli LPS (0.1–100 ng/mL) as controlRat neonatal brain microgliaEnhanced O2− generation, limited inflammatory mediator generation; MMP-9, macrophage inflammatory protein-2 (MIP-2/CXCL2) release, TXB2, concurrent with maximal O2− generation; elevated TXB2, MMP-9, tumor necrosis factor α (TNF-α), interleukin 1-α (IL-1α), and interleukin-6 (IL-6), macrophage inflammatory protein 1α (MIP-1α/CCL3), and MIP-2/CXCL2; LPS activates brain microglia in vitro and the release of O2−, inflammatory mediators[64]Microglia0.1–100 000 ng/mL Oscillatoria sp. LPS; 17 hRat neonatal microgliaClassical and alternative activation; pro-inflammatory and anti-inflammatory mediator release[65]MicrogliaScytonema javanicum and S. ocellatum LPSRat neonatal microgliaConcentration-dependent O₂-, MMP-9, IL-6 TNF-α, MIP-2/CXCL-2, CINC-1/CXCL-1, MIP-1α/CCL3, IL-10 release[66]Meningioma cells and meningioma–primary human macrophageSandwich immunoassayCyanobacterial LPS antagonist (CyP) 1–20μg/monolayerHuman meningioma cells and meningioma–primary human macrophage co-culturesCyanobacterial LPS inhibits cytokine production and augments the anti-inflammatory response when combined with benzylpenicillin[67]MicrogliaImmunocytochemical and immunofluorescent assay, ELISA, immunoblotting, live-cell imaging analysesCyanobacteria-derived TLR4 antagonist—a highly (95%) purified form of LPS-like molecule from Oscillatoria planktothrix sp. 20μg/mL for 24 h,Primary cultures from mouse spinal cordsTLR4 antagonists could be considered as a candidate of protective agents for motor neurons in degenerative diseases[68]Spleen cellsHot-water extract ofSpirulina platensisIn vitro cultures of murine spleen and thymus cellsIncreased proliferation of spleen cells; enhanced IL-1 production from peritoneal macrophages[69]hTHP-1ELISA, real-time PCRCyanobacterial LPS antagonist (CyP) fromOscillatoria planktothrix FP1; 10 µg/mL/5 hHuman THP-1 monocytic cell lineCyP is able to induce cross-tolerance toE. coli LPS by inhibiting TNF-α production[70]


## 2.7. Cytotoxicity of Lyngbyatoxins (Table7)

Lyngbyatoxins were first identified from Moorea producens (formerly Lyngbya majuscula). They are tumor-promoting agents which bound eucaryotic protein kinase C (PKC) isozymes [3].Table 7
Cytotoxicity of lyngbyatoxins.
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.FibroblastsMTT assay, [3H]-thymidine incorporation assay15 mg/mL (w/v) of the cyanobacterial extract/4 h or 24h for MTT test; 24 h for [3H]-thymidine incorporation assayPrimary mouse thymus fibroblasts80% inhibition of cell proliferation, morphology and attachment in 24 hDNA, cell membrane, cytoskeleton[70]FLNormal amniotic cells, humanStimulation of MTT reduction after 4h > 40% vs control cells; decreased cell viability to 32% of controls in 24 hA2058Human metastatic melanomaCytotoxic in 24 hRDHuman embryonic myosarcomaCytotoxic in 24 h3T3Mouse embryonic fibroblasts92% inhibition of cell proliferationL1210MTTLyngbyatoxin A and 12-epi-lyngbyatoxin A/18 hMouse lymphocytic leukemia cell lineCytotoxic effect; IC50 = 8.1 μM lyngbyatoxin A; IC50 = 20.4 μM 12-epi-lyngbyatoxin APKC isozymes[71]HL-60 CTest of induction of cell adhesionLyngbyatoxin A and debromoaplysia toxin/48 hHuman promyelocytic leukemia cellsFor 50% cell adhesion to the flasks—7 ng/mLLyngbyatoxin A and 700 ng/mL debromoaplysiatoxinCell membrane[72]DS 19Test of inhibition of terminal differentiationMouse erythroleukemia cells transformed by Friend leukemia virus strain 745AInhibition of terminal differentiation in 50% of the cells with 0.35 ng/mLLyngbyatoxin A and 150 ng/mL debromoaplysiatoxinNeuro-2aMTT24 hMouse neuroblastoma cellsCytotoxicity IC50 = 2.2 μM of hermitamides A; IC50 = 5.5 μM hermitamides B[73]CHOPatch-clamp0.1–30μM neo-debromoaplysia toxin G and HChinese hamster ovary cellsPotassium channel Kv1.5 block; IC50 = 1.79 μM debromo aplysiatoxin G and IC50 = 1.46 μM debromoaplysia toxin HVoltage-gated potassium channels Kv1.5 (KCNA5)[74]


## 2.8. Cytotoxicity of MCs (Table8)

MC are cyclic nonribosomal heptapeptides with low molecular weight (800–1100 Da), which contain several uncommon nonproteinogenic amino acids such as N-methyldehydroalanin (MDHA) derivatives and the uncommonβ-amino acid 3-amino-9-methoxy-2,6,8-trimethyldeca-4,6-dienoic acid (ADDA). MC are lipophilic toxins very resistant to hydrolysis, oxidation, and high temperatures. The main route of human exposure is the ingestion of contaminated drinking water, consumption of contaminated food or algal dietary supplements, and body contact, while more occasional routes are hemodialysis and inhalation. MC are classified mainly as hepatotoxins because they block eucaryotic PP (PP1, 2A and phosphoprotein phosphatases PPP4, PPP5) [2] through irreversible covalent binding [97]. Chronic and subchronic exposure to MC seems to be tumor promoting because they can increase the incidence of hepatic tumors in humans. MC could also enhance the oxidative stress. Additional target of MC in high concentrations is the ß-subunit of ATP synthase, causing mitochondrial apoptotic signaling. MC have hepatotoxic and tumor promoting action [3].Table 8
Cytotoxicity of MC.
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.CaCo-2Immuno-localization of MC uptakeMC-LR 1–75 µM/30 min–24 hImmortalized human colorectal adenocarcinoma cell lineArtificial epithelial cell layer is highly permeable to MC-LR[45]CaCo-2Gene expression, transcriptomicsMC-LR 10–100 µM/4–24 hThe sameOxidative stressERK/MAPK and cell cycle pathway molecules[75]CaCo-2Comet assay, MTT assay (for viability)MC-LR 0.2–10 µM/4–48 hThe same20% damaged DNA after 0.2 µM/4 h MC-LR; 40% reduced cell viability after MC-LR 10 µM/48 h,DNA[76]CaCo-2Protein phosphatase (PP) inhibition, LDH leakage, cell morphology and proliferation1–50 µM MC-LR, -LF and -LW for 22–48 hThe samePP inhibition—3.0 nM MC-LF, 3.8 nM MC-LW, 1.0 nM MC-LR, EC50 of LDH leakage: 25% (50 µM MC-LR), 36% (MC-LW), 51% (MC-LF), chromatin cell shrinkage, condensation, membrane blebbing, and cytoskeletal reorganizationPP, cell membrane, chromatin, cytoskeleton[77]CaCo-2Bradford assay, MTS reduction (for viability), neutral red uptakeMC-LR, –RR and -YR, 50–200 µM/24–48 hThe sameEC50 reduction of total protein content by MC-LR 111.1 ± 3 µM/24 h and MC-RR ˃200 µM/48 h; neutral red uptake—MC-YR 57.3 µM/48hProtein synthesis[78, 79]CaCo-2Immuno-localization of microcystinsMC-LR, –RR, 1–50 µM/30 min–24 hThe sameFacilitated MC uptake in <1 h by organic anion transporters, active excretionOrganic anion transporters 3A1 and 4A1[80]HIEC-6Cell counting Kit-8 for viability, western blot, TEER, PP2A activityMC-LR 0–50 µM/6–24 hHuman intestinal (colon) epithelial cell lineViability–12.5 µM/24 h; TEER at 50 µM/12 h and at 12.5 µM/24 h; apoptosis at 12.5 µM/24 h; western blot at 12.5 µM/24 h; occludin; claudin not affected), 25 µM/24 h; ZO-1; PP2A activity decreases from 12.5 µM/24 hPP2A, occludin, claudin[81]HEK293Western blot, luciferase assay, rtPCRMC-LR 10 µM/24 hHuman embryonic kidney cellsPP2A inhibition, enhanced proto-oncogene C-myc expressionPP2A, c-Myc protein, proto-oncogene C-myc[82]NCCPP2A, PP2B, PP2C activity, western blot, Akt, p38, JNK, PI3K assays, genechip analyses;MC-LR, 0.0001–1.0 µg/24 hImmortalized colorectal crypt cellsConstitute activation of Akt/p38 and JNK/MAPK pathwaysAkt, p38, JNK[83]HBE1, 16HBE14o-RT-PCR, western blot, RTCA, neutral red uptakeMC-LR 1–20 µM/48 hHuman bronchial epithelial cell linesNo effect on viability, ERK1/2 and p38 activities were not changedERK1/2 and p38 not influenced[9]DLD-1, HT-29Western blot, RT-qPCR, knockdown of SMAD2 by siRNA, migration and invasion assayMC-LR, 0.1–50 nM/24 hHuman colorectal cancer cellsInduction of SMAD2 signal transducer and transcriptional modulating protein expression, its activating phosphorylation by PI3K/Akt, increased migration (epithelial-mesenchymal transition of both cell types)PI3K/Akt, SMAD2,[84]BALB/cmRNAMC-LR 1–1000 nmol/L/6 hMouse peritoneal macrophagesDecreased transcription of mRNA for iNOS, IL‐1β, TNF‐α, GM‐CSF, and IFN‐γ; reduced inflammatory response to LPSiNOS, IL‐1β, TNF‐α, GM‐CSF and IFN‐γ[85]RAW 264.7 macrophagesWestern blot, ELISAMC-LR, 1–1000 nmol/L/30 min–24 hAbelson leukemia virus-transformed cell line from BALB/c miceActivation of NF-κB with 1000 nM and ERK1/2 with 100 nM; TNF-α synthesis (1 nM)NF-κB, ERK1/2, TNF-α[86]HepG2RT-qPCR, Western blot, MTT assay, mitochondrial membrane potential (MMP)MC-LR, 0.01–5 µM/3, 6, 12 and 24 hHuman hepatocellular carcinoma cell lineMMP loss, SOD induction in hypoxia, inhibitory apoptosis protein (c-IAP2) up-regulated in normoxic conditionMitochondrial dehydrogenase, SOD, c-IAP2[87]A549MTT assay, PP2A activity, Western blot, proliferationMC-LR, 0.5–10 µM/24 hHuman non-small-cells lung cancer cellsRearrangements of filamentous actin and microtubules due to PP2A/C (>1 µM) and p38 MAPK activation (0.5–10 µM); p-Blc-2, p-Bad (1.0–10 µM)Microtubules and filamentous actin (cytoskeleton), PP2A/C, p38,[88]HEK293Western blot, cell detachment, PP2A activity, MTT assayMC-LR, 0.5–10 µM/24 hHuman embryonic kidney cellsPP2A inhibition (>5 µM); PP2A activation (1–2 µM); cell anoikisPP2A catalytical and regulatory subunits[89]PC12Western blot, PP2A activity, immuno-fluorescenceMC-LR, 0.1–10 µM/6 hPheochromocytoma cells of the rat adrenal medullaRearrangement of filamentous actin and microtubules due to PP2A (>0.5 µM) and p38 MAPKPP2A, p38 MAPK, HSP27[90]HL7702PP2A activity, western blot, immuno-fluorescenceMC-LR, 5 or 10 µM for 30 min to 24 h;Human normal liver cell lineActivation of p38 MAPK, JNK and ERK1/2, HSP27-sensiitive cytoskeleton reassembly, PP2A inhibition in 6–24 h; activated phosphorylation of tau (by P38 MAPK) and VASPp38 MAPK, JNK, ERK1/2, PP2A; tau and VASP components of cytoskeleton[91, 92]SMMC-7721PP2A activity, western blot, PKA activity and Rac1/Cdc42 activity immuno-fluorescence, immuno-precipitationMC-LR, 0.5–10 µM/24 hHuman liver cancer cell linep-HSP27, p-VASP and p-cofilin contributed to cytoskeleton change; PP2A inhibition (>0.5 µM); disorder of cytoskeletonHSP27, VASP, cofilin, PKA, Rac1, PP2A[93]HepaRGCytopathic effects, RNA quantified by Agilent RNA 6000 Nano kitMC-LR, –RR 10, 100 and 1000 ng/2 hHuman hepatocyte cell lineIncrease of RNA of apoptotic and inflammatory gene; many cellular pathways activated[94]HL7702Real-time cell analyzer (RTCA) proliferation, cell cycle analysis, western blot, PP2A activity, MTT assay, immuno-fluorescenceMC-LR, 1, 5, 10 µM/1–96 hHuman normal liver cell lineMC-LR promoted HL7702 cell proliferation (36–48 h); activation of Akt/S6K1 cascade; PP2A activity (>1 µM), hyper-phosphorylation of Bcl-2, Bad, c-Myc and c-Jun, 1–10 µMPI3K/Akt/S6K1, hyper-phosphorylation of Bcl-2, Bad, c-Myc and c-Jun[95]HBEMTT and Annexin V/PI assay, ROS and MMP measurements, western blotMC-LR, 1, 10, 20, 30, 40 µg/mL/24, 48 hHuman bronchial epithelial cellsInducing mitochondria-dependent apoptosis (1–40 µg/mL), MMP decreases at 10 µg/mLCaspases[89]Huh7MC-LR, 0.5–50μM/6–72 hHuman hepatoma cells5μM MC-LR induced PP2A mRNA expression, p-CREB, expression of NF-κB, IFN-α, and several INFα-stimulated genes are activatedNF-κB, p-CREB, DNA[96]Abbreviations: CREB – cAMP responsive element-binding protein; ERK/MAPK – extracellular signal-regulated kinase/mitogen-activated protein kinase; GM‐CSF – granulocyte macrophage colony-stimulating factor; IFN-γ – interferon gamma; iNOS – inducible nitric oxide synthase; JNK–c – Jun N-terminal kinases; mRNA – messenger RNA; siRNA – small interfering RNA, VASP – vasodilator-stimulated phosphoprotein.


## 2.9. Cytotoxicity of Nodularins (Table9)

Nodularins (NODs) are cyclic nonribosomal pentapeptides and contain several unusual nonproteinogenic amino acids such as N-methyl-didehydroaminobutyric acid and theßβ-amino acid (all-S, all-E)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (ADDA). Ten variants have been discovered with nodularin-R being the predominant toxin variant. NODs are relatively stable compounds, with low sensitivity to light or temperature. NOD affects hepatocytes binding their PPs by noncovalent bonds, which increases the rate of phosphorylation. They are often attributed to gastroenteritis, allergic irritation reactions, and liver diseases. Nodularin-R is the most notorious as a potent hepatotoxin that may cause serious damage to the liver of humans and other animals. NODs have similar effects as microcystins and weak carcinogenicity [3].Table 9
Cytotoxicity of nodularins.
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.CLCFluorometric cell membrane integrity, cell viability and ROS measurements, caspase-glo 3/7 assay, ELISANOD, 0.001, 0.01, 0.05, 0.1μg/mL/24 hCarp leukocyte cell line and head kidney leukocytesCell viability, membrane integrity at 0.1μg/mL, DNA fragmentation and caspases 3/7 activation at > 0.1 μg/mL, ROS increase in 60 min in >0.01 μg/mL, GSH decrease at >0.001/24 hGSH/GSSG, DNA, membranes, caspases[98]CLC and kidney leucocytesFluorometric cell viability, ROS and nitrogen species (NS) measurements0.001, 0.01, 0.05, or 0.1μg/mL/24 hCarp leukocyte cell line, kidney leukocytesCytotoxicity ≥0.05μg/mL, ROS and NS increase, expression of TNF-α, IL-10, less TGF-βDNA expression[99]HepG2Micronucleus assay, Flow cytometry, comet assay, DNA damageNOD, 1–10μg/mL, for 6, 12, 24, 48 hHuman hepatoma cell lineDNA damage >1μg/mL, apoptosis from1 μg/mL/48 hDNA, cellular and mitochondrial membranes[100]HepG2RT-PCR, siRNA, flow cytometry, transfection of NF-κB immunoblotingNOD, 2.5, 5, 7.5, 10μM/24 hHuman hepatoma cell lineInduces fas receptor (fas) and fas ligand (FasL) expression and apoptosisNF-κB pathway, fas, FasL[101]HepG2 and Huh7ATF-6 activity qPCR, TNF-α ELISA, immunoblottingNOD, 0.1, 1, 5μM for 24, 48 and 72 hHuman hepatoma cell linesInduction of TNF-α protein, CAAT/enhancer-binding protein-homologous,TNF-α, ERK 1/2 MAPK, ER, IL-8, CHOP ER-stress—2.5 nM[102]HepG2qPCR, MTT assay, comet assay, cytokinesis micronucleus assayNOD, 0.01, 0.1 and 1μg/mLHuman hepatoma cell lineDNA damage; apoptosis (BAX, BCL2) genes, ROS increase, oxidative stressDNA, ROS[103]


## 2.10. Cytotoxicity of Retinoids from Cyanobacteria (Table10)

Retinol, a novel retinoic acid (RA) analogue 7-hydroxy RA, 4-oxo-RA, and several analogues were identified in cyanobacterial blooms [110]. They act as RA receptors that may cause different malformations, as well as to have a teratogenic effect on aqueous animals.Table 10
Cytotoxicity of cyanobacterial retinoids.
Cell typeAssayConditionsTissue of originMain effectsTargetsRef.P19/A15Bioluminescence reporter assay; calcein AM cell viability assay0.25, 0.5, 1 and 2 g dm/l/24 h; water extracts 2.5x–20x environmental water/24hMurine embryonal carcinoma cells stably transfected with firefly luciferase geneRetinoid-like activity, max 263 ng retinoid eq/L; cytotoxic effect at 20xRAR[104]P19/A15Bioluminescence reporter assay;0.25, 0.5, 1 and 2 g dw/l/24hMurine embryonal carcinoma cellsRetinoid-like activityRAR[105]P19/A15RAR/RXR transactivation assayCyanobacterial extracts 0.125–2 g dw/l/24 hMurine embryonal carcinoma cellsRetinoid acid receptor (RAR) activityRAR[106]P19/A15Bioluminescence reporter assayCyanobacterial extracts 0.25–2 g dm/l and exudates 2.5×–20×/24 hMurine embryonal carcinoma cellsRetinoid acid receptor activityRAR[107]P19/A15Bioluminescence reporter assay1x–20x concentrated cyanobacterial and algal exudates/24hMurine embryonal carcinoma cellsRetinoid-like activityRAR[108]HepG2MTT, comet assay, cytokinesis-block micronucleus (cytome) assay0.04–2 mg/mL/24h for MTT, 0.2 mg dm/mL/24h for cytokinesis-block micronucleus assayHuman hepatocellular carcinoma cellsSignificant genotoxic effects of retinoic acid from the extractsDNA[109]


## 2.11. Cytotoxicity of saxitoxins (Table11)

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. They have a neurotoxic effect by blocking voltage-gated sodium channels [3].Table 11
Cytotoxicity of SXT.
Cell typeAssayConditionsTissue of originMain effectsRef.Neuro-2AHPLC, LC-MS/MS, Jellett rapid test, MTT assaySTX, 0.05–200 ng/mLMouse neuroblastoma cell lineScreening assay for determination of toxicity and comparison of various methods for detection of toxins[111]IEC-6, Caco-2HPLC,Gonyautoxin, 100μM for 1–60 minHuman colorectal, adenocarcinoma cell lineIEC-6 cells secrete the toxin, Caco-2 cells absorb it Na+-dependently[112]


## 3. Limitations

Studies on cell cultures cannot reveal all possible effects of toxins on the human body. This is due to the following reasons: (1) no matter how many cultures are tested, they will not cover the whole variety of cells in the body; (2) there are often significant differences between the cells in culture, the primary cell lines and the cells in the body tissues in the quantity and quality of expressed proteins (genes expression), metabolic pathways and cell function [113–115]. Therefore, results from cells in culture cannot be directly transferred to the tissue of origin or of which they will form. (3) Numerous regulations are active continuously and simultaneously in the organism, and their cross-influence cannot be simulated in experiments with cell cultures. (4) Parameters like LC50 or ID50 are different for cells in culture and human body.


## 4. Perspectives

The 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. The use of cell cocultures [116–118] 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.


## 5. Conclusion

The 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. Therefore, 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.



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*Source: 5647178-2022-04-25.xml*