Microcystin-LR (MC-LR) is a potent hepatotoxin produced by harmful cyanobacterial blooms (CyanoHABs). MC-LR targets highly differentiated hepatocytes expressing organic anion transporting polypeptides OATP1B1 and OATP1B3 that are responsible for hepatocellular uptake of the toxin. The present study utilized an advanced 3D in vitro human liver model Hepoid-HepaRG based on the cultivation of collagen-matrix embedded multicellular spheroids composed of highly differentiated and polarized hepatocyte-like cells. 14-d-old Hepoid-HepaRG cultures showed increased expression of OATP1B1/1B3 and sensitivity to MC-LR cytotoxicity at concentrations >10 nM (48 h exposure, EC20 = 26 nM). MC-LR induced neither caspase 3/7 activity nor expression of the endoplasmic reticulum stress marker gene BiP/GRP78, but increased release of pro-inflammatory cytokine IL-8, indicating a necrotic type of cell death. Subcytotoxic (10 nM) and cytotoxic (≥100 nM) MC-LR concentrations disrupted hepatocyte functions, such as xenobiotic metabolism phase-I enzyme activities (cytochrome P450 1A/1B) and albumin secretion, along with reduced expression of CYP1A2 and ALB genes. MC-LR also decreased expression of HNF4A gene, a critical regulator of hepatocyte differentiation and function. Genes encoding hepatobiliary membrane transporters (OATP1B1, BSEP, NTCP), hepatocyte gap junctional gene connexin 32 and the epithelial cell marker E-cadherin were also downregulated. Simultaneous upregulation of connexin 43 gene, primarily expressed by liver progenitor and non-parenchymal cells, indicated a disruption of tissue homeostasis. This was associated with a shift in the expression ratio of E-cadherin to N-cadherin towards the mesenchymal cell marker, a process linked to epithelial-mesenchymal transition (EMT) and hepatocarcinogenesis. The effects observed in the human liver cell in vitro model revealed mechanisms that can potentially contribute to the MC-LR-induced promotion and progression of hepatocellular carcinoma (HCC). Hepoid-HepaRG cultures provide a robust, accessible and versatile in vitro model, capable of sensitively detecting hepatotoxic effects at toxicologically relevant concentrations, allowing for assessing hepatotoxicity mechanisms, human health hazards and impacts of environmental hepatotoxins, such as MC-LR.

• Klíčová slova
• 3D human liver model Hepoid-HepaRG, Cyanotoxins, Hepatocarcinogenesis, Microcystin-LR, Organic anion transporting polypeptides,
• MeSH
• hepatocelulární karcinom * MeSH
• kadheriny MeSH
• lékové postižení jater * MeSH
• lidé MeSH
• mikrocystiny toxicita   metabolismus   MeSH
• mořské toxiny * MeSH
• nádory jater * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cyanoginosin LR MeSH Prohlížeč
• kadheriny MeSH
• mikrocystiny MeSH
• mořské toxiny * MeSH

Components of cyanobacterial water blooms were quantified in aerosols above agitated water surfaces of five freshwater bodies. The thoracic and respirable aerosol fraction (0.1-10 µm) was sampled using a high-volume sampler. Cyanotoxins microcystins were detected by LC-MS/MS at levels 0.3-13.5 ng/mL (water) and < 35-415 fg/m3 (aerosol). Lipopolysaccharides (endotoxins) were quantified by Pyrogene rFC assay at levels < 10-119 EU/mL (water) and 0.13-0.64 EU/m3 (aerosol). Cyanobacterial DNA was detected by qPCR at concentrations corresponding to 104-105 cells eq./mL (water) and 101-103 cells eq./m3 (aerosol). Lipopolysaccharides isolated from bloom samples induced IL-6 and IL-8 cytokine release in human bronchial epithelial cells Beas-2B, while extracted cyanobacterial metabolites induced both pro-inflammatory and cytotoxic effects. Bloom components detected in aerosols and their bioactivities observed in upper respiratory airway epithelial cells together indicate that aerosols formed during cyanobacterial water blooms could induce respiratory irritation and inflammatory injuries, and thus present an inhalation health risk.

• Klíčová slova
• Aerosol, Cyanobacteria, Cyanotoxins, Inflammation, Inhalation toxicity, Lipopolysaccharides,
• MeSH
• aerosoly MeSH
• chromatografie kapalinová MeSH
• lidé MeSH
• lipopolysacharidy analýza   MeSH
• mikrocystiny toxicita   MeSH
• sinice * metabolismus   MeSH
• sladká voda analýza   MeSH
• tandemová hmotnostní spektrometrie MeSH
• toxiny kmene Cyanobacteria * MeSH
• voda MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aerosoly MeSH
• lipopolysacharidy MeSH
• mikrocystiny MeSH
• toxiny kmene Cyanobacteria * MeSH
• voda MeSH

Fish are exposed to numerous stressors in the environment including pollution, bacterial and viral agents, and toxic substances. Our study with common carps leveraged an integrated approach (i.e., histology, biochemical and hematological measurements, and analytical chemistry) to understand how cyanobacteria interfere with the impact of a model viral agent, Carp sprivivirus (SVCV), on fish. In addition to the specific effects of a single stressor (SVCV or cyanobacteria), the combination of both stressors worsens markers related to the immune system and liver health. Solely combined exposure resulted in the rise in the production of immunoglobulins, changes in glucose and cholesterol levels, and an elevated marker of impaired liver, alanine aminotransferase (ALT). Analytical determination of the cyanobacterial toxin microcystin-LR (MC-LR) and its structurally similar congener MC-RR and their conjugates showed that SVCV affects neither the levels of MC in the liver nor the detoxification capacity of the liver. MC-LR and MC-RR were depurated from liver mostly in the form of cysteine conjugates (MC-LR-Cys, MC-RR-Cys) in comparison to glutathione conjugates (LR-GSH, RR-GSH). Our study brought new evidence that cyanobacteria worsen the effect of viral agents. Such inclusion of multiple stressor concept helps us to understand how and to what extent the relevant environmental stressors co-influence the health of the fish population.

• Klíčová slova
• conjugates, cyanobacteria, immune system, microcystins, spring viraemia of carp,
• MeSH
• chemické látky znečišťující vodu toxicita   MeSH
• kapři mikrobiologie   MeSH
• Microcystis chemie   MeSH
• mikrocystiny toxicita   MeSH
• nemoci ryb chemicky indukované   patofyziologie   MeSH
• roční období MeSH
• stupeň závažnosti nemoci * MeSH
• testy toxicity MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chemické látky znečišťující vodu MeSH
• mikrocystiny MeSH

Various stressors including temperature, environmental chemicals, and toxins can have profound impacts on immunity to pathogens. Increased eutrophication near rivers and lakes coupled with climate change are predicted to lead to increased algal blooms. Currently, the effects of cyanobacterial toxins on disease resistance in mammals is a largely unexplored area of research. Recent studies have suggested that freshwater cyanotoxins can elicit immunomodulation through interaction with specific components of innate immunity, thus potentially altering disease susceptibility parameters for fish, wildlife, and human health owing to the conserved nature of the vertebrate immune system. In this study, we investigated the effects of three microcystin congeners (LR, LA, and RR), nodularin-R, and cylindrospermopsin for their ability to directly interact with nine different human Toll-like receptors (TLRs)-key pathogen recognition receptors for innate immunity. Toxin concentrations were verified by LC/MS/MS prior to use. Using an established HEK293-hTLR NF-κB reporter assay, we concluded that none of the tested toxins (29-90 nM final concentration) directly interacted with human TLRs in either an agonistic or antagonistic manner. These results suggest that earlier reports of cyanotoxin-induced NF-κB responses likely occur through different surface receptors to mediate inflammation.

• Klíčová slova
• Cyanotoxin, Inflammation, NF-κB, Toll-like receptor,
• MeSH
• alkaloidy MeSH
• cyklické peptidy MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mikrocystiny * toxicita   MeSH
• tandemová hmotnostní spektrometrie * MeSH
• toll-like receptory genetika   MeSH
• toxiny kmene Cyanobacteria MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alkaloidy MeSH
• cyklické peptidy MeSH
• cylindrospermopsin MeSH Prohlížeč
• mikrocystiny * MeSH
• nodularin MeSH Prohlížeč
• toll-like receptory MeSH
• toxiny kmene Cyanobacteria MeSH

Secondary metabolites of cyanobacteria and algae released during algal blooms often exhibit toxic effects, but only a small number of the metabolites are the subject of routine analytical screenings. Alternatively, ecotoxicological assays offer a better representation of the overall negative effects. The aim of this work was to compare multiple assays in their sensitivity towards cellular algal organic matter (COM) of the toxin-producing cyanobacterium Microcystis aeruginosa. Multiple endpoints were investigated: mortality, growth inhibition, bioluminescence inhibition, genotoxicity, endocrine-disrupting effects, oxidative stress, and the induction of ethoxyresorufin-O-deethylase (EROD). Three rainbow trout (Oncorhynchus mykiss) cell lines as well as representatives of bacteria, yeasts, algae, vascular plants, and crustaceans were employed, and the results were expressed per mg of dissolved organic carbon (DOC) in the COM. M. aeruginosa COM was toxic to the RTgill-W1, RTG-2, and RTL-W1 cell lines (EC50 values ranging from 0.48 ± 0.02 to 1.9 ± 0.1 mgDOC/L), to the crustacean Thamnocephalus platyurus (LC50 = 20 ± 1 mgDOC/L), and to Lepidium sativum (IC50 = 241 ± 13 mgDOC/L). In contrast, no effect was observed for bacteria and yeasts, and the growth of the alga Desmodesmus subspicatus was even stimulated. No genotoxicity, endocrine-disrupting effects or increase in oxidative stress or EROD activity was detected. The content of six microcystins (MC-LR, MC-RR, MC-YR, MC-LY, MC-LW, and MC-LF), anatoxin-a, cylindrospermopsin, and nodularin in the M. aeruginosa COM was determined by liquid chromatography-tandem mass spectrometry. An artificially prepared mixture of the detected cyanotoxins in the corresponding concentrations did not induce response in the O. mykiss cell lines and T. platyurus, suggesting that other cyanobacterial metabolites are responsible for the toxicity of M. aeruginosa.

• Klíčová slova
• Cellular algal organic matter, Cytotoxicity, Harmful algal blooms, Microcystin, Microcystis aeruginosa, Rainbow trout cell lines,
• MeSH
• eutrofizace MeSH
• Microcystis * MeSH
• mikrocystiny toxicita   MeSH
• sinice * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mikrocystiny MeSH

In the last decade, it has become evident that complex mixtures of cyanobacterial bioactive substances, simultaneously present in blooms, often exert adverse effects that are different from those of pure cyanotoxins, and awareness has been raised on the importance of studying complex mixtures and chemical interactions. We aimed to investigate cytotoxic and genotoxic effects of complex extracts from laboratory cultures of cyanobacterial species from different orders (Cylindrospermopsis raciborskii, Aphanizomenon gracile, Microcystis aeruginosa, M. viridis, M. ichtyoblabe, Planktothrix agardhii, Limnothrix redekei) and algae (Desmodesmus quadricauda), and examine possible relationships between the observed effects and toxin and retinoic acid (RA) content in the extracts. The cytotoxic and genotoxic effects of the extracts were studied in the human hepatocellular carcinoma HepG2 cell line, using the MTT assay, and the comet and cytokinesis-block micronucleus (cytome) assays, respectively. Liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) was used to detect toxins (microcystins (MC-LR, MC-RR, MC-YR) and cylindrospermopsin) and RAs (ATRA and 9cis-RA) in the extracts. Six out of eight extracts were cytotoxic (0.04-2 mgDM/mL), and five induced DNA strand breaks at non-cytotoxic concentrations (0.2-2 mgDM/mL). The extracts with genotoxic activity also had the highest content of RAs and there was a linear association between RA content and genotoxicity, indicating their possible involvement; however further research is needed to identify and confirm the compounds involved and to elucidate possible genotoxic effects of RAs.

• Klíčová slova
• algae, chemical analysis, complex mixtures, cyanobacteria, cyanotoxins, cytotoxicity, extracts, genotoxicity, microcystins, retinoic acids,
• MeSH
• alkaloidy izolace a purifikace   toxicita   MeSH
• buňky Hep G2 MeSH
• Chlorophyta metabolismus   MeSH
• chromatografie kapalinová MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• kometový test MeSH
• lidé MeSH
• mikrocystiny izolace a purifikace   toxicita   MeSH
• mikrojaderné testy MeSH
• mikrojádra chromozomálně defektní chemicky indukované   MeSH
• poškození DNA * MeSH
• sinice metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• toxiny kmene Cyanobacteria MeSH
• tretinoin izolace a purifikace   toxicita   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkaloidy MeSH
• cylindrospermopsin MeSH Prohlížeč
• microcystin MeSH Prohlížeč
• mikrocystiny MeSH
• toxiny kmene Cyanobacteria MeSH
• tretinoin MeSH

Changes in ecological and environmental factors lead to an increased occurrence of cyanobacterial water blooms, while secondary metabolites-producing cyanobacteria pose a threat to both environmental and human health. Apart from oral and dermal exposure, humans may be exposed via inhalation and/or swallowing of contaminated water and aerosols. Although many studies deal with liver toxicity, less information about the effects in the respiratory system is available. We investigated the effects of a prevalent cyanotoxin, microcystin-LR (MC-LR), using respiratory system-relevant human bronchial epithelial (HBE) cells. The expression of specific organic-anion-transporting polypeptides was evaluated, and the western blot analysis revealed the formation and accumulation of MC-LR protein adducts in exposed cells. However, MC-LR up to 20 μM neither caused significant cytotoxic effects according to multiple viability endpoints after 48-h exposure, nor reduced impedance (cell layer integrity) over 96 h. Time-dependent increase of putative MC-LR adducts with protein phosphatases was not associated with activation of mitogen-activated protein kinases ERK1/2 and p38 during 48-h exposure in HBE cells. Future studies addressing human health risks associated with inhalation of toxic cyanobacteria and cyanotoxins should focus on complex environmental samples of cyanobacterial blooms and alterations of additional non-cytotoxic endpoints while adopting more advanced in vitro models.

• Klíčová slova
• 16HBE14o-, mitogen-activated protein kinase, HBE1, OATP, cytotoxicity, human bronchial epithelial cells, in vitro, microcystin-LR,
• MeSH
• bronchy cytologie   MeSH
• buněčné linie MeSH
• epitelové buňky účinky léků   metabolismus   MeSH
• extracelulárním signálem regulované MAP kinasy metabolismus   MeSH
• lidé MeSH
• mikrocystiny toxicita   MeSH
• mitogenem aktivované proteinkinasy p38 metabolismus   MeSH
• mořské toxiny toxicita   MeSH
• přenašeče organických aniontů genetika   MeSH
• signální transdukce účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyanoginosin LR MeSH Prohlížeč
• extracelulárním signálem regulované MAP kinasy MeSH
• mikrocystiny MeSH
• mitogenem aktivované proteinkinasy p38 MeSH
• mořské toxiny MeSH
• přenašeče organických aniontů MeSH

As human co-exposure to natural toxins through food and water is inevitable, risk assessments to safeguard health are necessary. Aflatoxin B1 and fumonisin B1, frequent co-contaminants of maize and microcystin-LR, produced in freshwater by cyanobacteria are all naturally occurring potent toxins that threaten human health. Populations in the poorest regions of the world may suffer repeated simultaneous exposure to these contaminants. Using High Content Analysis, multiple cytotoxicity endpoints were measured for the individual toxins and mixtures in various cell lines. Results highlighted that significant cytotoxic effects were observed for aflatoxin B1 in all cell lines while no cytotoxic effects were observed for fumonisin B1 or microcystin-LR. Aflatoxin B1/microcystin-LR was cytotoxic in the order HepG2 > Caco-2 > MDBK. Fumonisin B1/microcystin-LR affected MDBK cells. The ternary mixture was cytotoxic to all cell lines. Most combinations were additive, however antagonism was observed for binary and ternary mixtures in HepG2 and MDBK cell lines at low and high concentrations. Synergy was observed in all cell lines, including at low concentrations. The combination of these natural toxins may pose a significant risk to populations in less developed countries. Furthermore, the study highlights the complexity around trying to regulate for human exposure to multiple contaminants.

• Klíčová slova
• Aflatoxin B(1), Combined exposure, Cytotoxicity, Fumonisin B(1), High content analysis, Microcystin-LR,
• MeSH
• aflatoxin B1 aplikace a dávkování   chemie   toxicita   MeSH
• biologické markery moč   MeSH
• biologické toxiny MeSH
• buněčné linie MeSH
• fumonisiny aplikace a dávkování   chemie   toxicita   MeSH
• kontaminace potravin MeSH
• lidé MeSH
• mikrocystiny aplikace a dávkování   chemie   toxicita   MeSH
• mořské toxiny MeSH
• skot MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aflatoxin B1 MeSH
• biologické markery MeSH
• biologické toxiny MeSH
• cyanoginosin LR MeSH Prohlížeč
• fumonisin B1 MeSH Prohlížeč
• fumonisiny MeSH
• mikrocystiny MeSH
• mořské toxiny MeSH

Cyanobacteria produce many biologically active metabolites synthesized via nonribosomal synthetic pathways such as cyclic microcystins (MCs) and linear aeruginosins (Aers). The present study aimed to investigate the effects of different MC variants and the newly isolated aerugenosin Aer-865 on macrophages, which represent one of the key effector cells within the innate immune responses. Specifically, our study included RAW 264.7 macrophage activation associated with production of cytotoxic and cytostatic nitric oxide (NO) as well as pro-inflammatory mediators like tumor necrosis factor α (TNFα) and interleukin 6 (IL-6). From the compounds investigated, commonly occurring MC variants (-RR, -YR) and Aer-865 had no significant effects within the non-cytotoxic concentrations tested, i.e. 0.001-1 μM for MCs and 0.1-50 μM for Aer-865. In contrast to known immunoactive MC-LR, the negligible immunomodulatory potential of tested MC congeners could be related to their differences in structure. The knowledge of MC structure-specific activities contributes to the understanding of complex toxicity of different MC variants and most importantly their mixtures. This study is one of the first study that evaluate the effect of larger set of cyanobacterial peptides on macrophages and compare their immunomodulatory potential.

• Klíčová slova
• Cyanobacteria, Cyanotoxins, Innate immunity, Macrophages,
• MeSH
• bakteriální toxiny chemie   toxicita   MeSH
• bakteriociny chemie   toxicita   MeSH
• imunomodulace účinky léků   MeSH
• makrofágy účinky léků   imunologie   MeSH
• mikrocystiny chemie   toxicita   MeSH
• mořské toxiny chemie   toxicita   MeSH
• myši MeSH
• RAW 264.7 buňky MeSH
• toxiny kmene Cyanobacteria MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aeruginocin MeSH Prohlížeč
• bakteriální toxiny MeSH
• bakteriociny MeSH
• mikrocystiny MeSH
• mořské toxiny MeSH
• toxiny kmene Cyanobacteria MeSH

HL1-hT1 cell line represents adult human liver stem cells (LSCs) immortalized with human telomerase reverse transcriptase. In this study, HL1-hT1 cells were found to express mesenchymal markers (vimentin, CD73, CD90/THY-1 and CD105) and an early hepatic endoderm marker FOXA2, while not expressing hepatic progenitor (HNF4A, LGR5, α-fetoprotein) or differentiated hepatocyte markers (albumin, transthyretin, connexin 32). In response to microcystin-LR (MC-LR), a time- and concentration-dependent formation of MC-positive protein bands in HL1-hT1 cells was observed. Cellular accumulation of MC-LR occurred most likely via mechanisms independent on organic anion transporting polypeptides (OATPs) or multidrug resistance (MDR) proteins, as indicated (a) by a gene expression analysis of 11 human OATP genes and 4 major MDR genes (MDR1/P-glycoprotein, MRP1, MRP2 and BCRP); (b) by non-significant effects of OATP or MDR1 inhibitors on MC-LR uptake. Accumulation of MC-positive protein bands in HL1-hT1 cells was associated neither with alterations of cell viability and growth, dysregulations of ERK1/2 and p38 kinases, reactive oxygen species formation, induction of double-stranded DNA breaks nor modulations of stress-inducible genes (ATF3, HSP5). It suggests that LSCs might have a selective, MDR1-independent, survival advantage and higher tolerance towards MC-induced cytotoxic, genotoxic or cancer-related events than differentiated adult hepatocytes, fetal hepatocyte or malignant liver cell lines. HL1-hT1 cells provide a valuable in vitro tool for studying effects of toxicants and pharmaceuticals on LSCs, whose important role in the development of chronic toxicities and liver diseases is being increasingly recognized.

• Klíčová slova
• Adult liver stem cells, HL1-hT1, Liver tumor-promotion, Microcystin-LR, Multidrug resistance proteins, OATP,
• MeSH
• apoptóza účinky léků   fyziologie   MeSH
• dospělé kmenové buňky účinky léků   metabolismus   MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• inhibitory enzymů toxicita   MeSH
• játra cytologie   účinky léků   metabolismus   MeSH
• karcinogeny toxicita   MeSH
• lidé MeSH
• mikrocystiny toxicita   MeSH
• mořské toxiny MeSH
• transformované buněčné linie MeSH
• viabilita buněk účinky léků   fyziologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyanoginosin LR MeSH Prohlížeč
• inhibitory enzymů MeSH
• karcinogeny MeSH
• mikrocystiny MeSH
• mořské toxiny MeSH