Cyanobacterial blooms are increasing in frequency and intensity globally, and impacting recreational waters as well as waters used for drinking water provisioning. They are sources of bioactive metabolites including retinoids and the neurotoxin anatoxin-a. Here, we investigated the effects of anatoxin-a on a differentiating in vitro human neural stem cell model previously characterised with retinoic acids. Effects on protein and gene expression upon exposure for 9 or 18 days to anatoxin-a alone or in co-exposure with all-trans retinoic acid were evaluated using a panel of neural and glial differentiation biomarkers. Anatoxin-a did not cause distinct developmental neurotoxicity alone, or in co-exposure with retinoic acid. However, in line with its excitotoxicity, in co-exposure with 200 nM all-trans retinoic acid it reduced the differentiation of acetylcholinergic neuron subtypes in the culture at 1000 nM (highest tested concentration). While this could have substantial functional implications for the developing nervous system, there is no indication for developmental neurotoxicity beyond its (excito-)toxicity to acetylcholinergic neurons, which only occurred in co-exposure to all-trans retinoic acid.

• MeSH
• exprese genu MeSH
• lidé MeSH
• neurotoxické syndromy * etiologie   MeSH
• retinoidy metabolismus   MeSH
• sinice * MeSH
• toxiny kmene Cyanobacteria MeSH
• tretinoin toxicita   MeSH
• tropany * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Cyanobacterial blooms are known sources of environmentally-occurring retinoid compounds, including all-trans and 9-cis retinoic acids (RAs). The developmental hazard for aquatic organisms has been described, while the implications for human health hazard assessment are not yet sufficiently characterized. Here, we employ a human neural stem cell model that can differentiate in vitro into a mixed culture of neurons and glia. Cells were exposed to non-cytotoxic 8-1000 nM all-trans or 9-cis RA for 9-18 days (DIV13 and DIV22, respectively). Impact on biomarkers was analyzed on gene expression (RT-qPCR) and protein level (western blot and proteomics) at both time points; network patterning (immunofluorescence) on DIV22. RA exposure significantly concentration-dependently increased gene expression of retinoic acid receptors and the metabolizing enzyme CYP26A1, confirming the chemical-specific response of the model. Expression of thyroid hormone signaling-related genes remained mostly unchanged. Markers of neural progenitors/stem cells (PAX6, SOX1, SOX2, NESTIN) were decreased with increasing RA concentrations, though a basal population remained. Neural markers (DCX, TUJ1, MAP2, NeuN, SYP) remained unchanged or were decreased at high concentrations (200-1000 nM). Conversely, (astro-)glial marker S100β was increased concentration-dependently on DIV22. Together, the biomarker analysis indicates an RA-dependent promotion of glial cell fates over neural differentiation, despite the increased abundance of neural protein biomarkers during differentiation. Interestingly, RA exposure induced substantial changes to the cell culture morphology: while low concentrations resulted in a network-like differentiation pattern, high concentrations (200-1000 nM RA) almost completely prevented such network patterning. After functional confirmation for implications in network function, such morphological features could present a proxy for network formation assessment, an apical key event in (neuro-)developmental Adverse Outcome Pathways. The described application of a human in vitro model for (developmental) neurotoxicity to emerging environmentally-relevant retinoids contributes to the evidence-base for the use of differentiating human in vitro models for human health hazard and risk assessment.

• MeSH
• alitretinoin * toxicita   MeSH
• buněčná diferenciace MeSH
• lidé MeSH
• nervové kmenové buňky * účinky léků   MeSH
• receptory kyseliny retinové genetika   metabolismus   MeSH
• retinoidy farmakologie   MeSH
• tretinoin * toxicita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cylindrospermopsin (CYN) is a cyanobacterial toxin that occurs in aquatic environments worldwide. It is known for its delayed effects in animals and humans such as inhibition of protein synthesis or genotoxicity. The molecular targets and the cell physiological mechanisms of CYN, however, are not well studied. As inhalation of CYN-containing aerosols has been identified as a relevant route of CYN uptake, we analyzed the effects of CYN on protein expression in cultures of immortalized human bronchial epithelial cells (16HBE14o-) using a proteomic approach. Proteins whose expression levels were affected by CYN belonged to several functional clusters, mainly regulation of protein stability, cellular adhesion and integration in the extracellular matrix, cell proliferation, cell cycle regulation, and completion of cytokinesis. With a few exceptions of upregulated proteins (e.g., ITI inhibitor of serine endopeptidases and mRNA stabilizer PABPC1), CYN mediated the downregulation of many proteins. Among these, centrosomal protein 55 (CEP55) and osteonectin (SPARC) were significantly reduced in their abundance. Results of the detailed semi-quantitative Western blot analyses of SPARC, claudin-6, and CEP55 supported the findings from the proteomic study that epithelial cell adhesion, attenuation of cell proliferation, delayed completion of mitosis, as well as induction of genomic instability are major effects of CYN in eukaryotic cells.

• MeSH
• epitelové buňky * účinky léků   metabolismus   MeSH
• lidé MeSH
• proteiny buněčného cyklu MeSH
• proteomika MeSH
• toxiny kmene Cyanobacteria * toxicita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

To generate a hepatitis E virus (HEV) genotype 3 (HEV-3)-specific monoclonal antibody (mAb), the Escherichia coli-expressed carboxy-terminal part of its capsid protein was used to immunise BALB/c mice. The immunisation resulted in the induction of HEV-specific antibodies of high titre. The mAb G117-AA4 of IgG1 isotype was obtained showing a strong reactivity with the homologous E. coli, but also yeast-expressed capsid protein of HEV-3. The mAb strongly cross-reacted with ratHEV capsid protein derivatives produced in both expression systems and weaker with an E. coli-expressed batHEV capsid protein fragment. In addition, the mAb reacted with capsid protein derivatives of genotypes HEV-2 and HEV-4 and common vole hepatitis E virus (cvHEV), produced by the cell-free synthesis in Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cell lysates. Western blot and line blot reactivity of the mAb with capsid protein derivatives of HEV-1 to HEV-4, cvHEV, ratHEV and batHEV suggested a linear epitope. Use of truncated derivatives of ratHEV capsid protein in ELISA, Western blot, and a Pepscan analysis allowed to map the epitope within a partially surface-exposed region with the amino acid sequence LYTSV. The mAb was also shown to bind to human patient-derived HEV-3 from infected cell culture and to hare HEV-3 and camel HEV-7 capsid proteins from transfected cells by immunofluorescence assay. The novel mAb may serve as a useful tool for further investigations on the pathogenesis of HEV infections and might be used for diagnostic purposes. KEY POINTS: • The antibody showed cross-reactivity with capsid proteins of different hepeviruses. • The linear epitope of the antibody was mapped in a partially surface-exposed region. • The antibody detected native HEV-3 antigen in infected mammalian cells.

• MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• Escherichia coli MeSH
• kapsida MeSH
• křečci praví MeSH
• lidé MeSH
• monoklonální protilátky MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• virové plášťové proteiny MeSH
• virus hepatitidy E * MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články 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.

• 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

Anthropogenic eutrophication of freshwater bodies increases the occurrence of toxic cyanobacterial blooms. The cyanobacterial toxin cylindrospermopsin (CYN) is detected in the environment with increasing frequency, driving the scientific effort to assess emerging health risks from CYN-producing blooms. Oral exposure to CYN results primarily in hepatotoxicity. Nevertheless, extrahepatic manifestations of CYN toxicity have been reported. Furthermore, cyanotoxins have been detected in aerosols and dust particles, suggesting potential toxic effects in the respiratory tract. To assess the susceptibility of airway epithelia towards cyanotoxins, monolayers of immortalized human bronchial epithelial cells HBE1 and 16HBE14o- were exposed to a concentration range of 0.1-10 μM CYN. Cytotoxic endpoints were assessed as morphologic alterations, resazurin reduction capacity, esterase activity, neutral red uptake, and by impedimetric real-time cell analysis. Depending on the endpoint assessed, EC50 values ranged between 0.7 and 1.8 μM (HBE1) and 1.6-4.8 μM (16HBE14o-). To evaluate alterations of other cellular events by subcytotoxic concentration of CYN (1 μM), phosphorylation of mitogen-activated protein kinases ERK and p38 was determined. Only a slight increase in p38 phosphorylation was induced by CYN in HBE1 cell line after 48 h, while activities of both ERK1/2 and p38 gradually and significantly increased in 16HBE14o- cells during 8-48 h exposure. This study suggests possible hazards of inhalation CYN exposures, which may severely impact the integrity of airway epithelia and epithelial cell signaling. Further research of CYN-induced toxicity and underlying mechanisms is needed, as well as more data on environmental concentrations of cyanotoxins in aerosols for exposure assessment.

• MeSH
• bakteriální toxiny farmakologie   MeSH
• buněčné linie MeSH
• dýchací soustava cytologie   MeSH
• epitelové buňky účinky léků   MeSH
• eutrofizace * MeSH
• lidé MeSH
• mikrocystiny farmakologie   MeSH
• mořské toxiny farmakologie   MeSH
• uracil analogy a deriváty   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH