Effects of all-trans and 9-cis retinoic acid on differentiating human neural stem cells in vitro.

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 S100ß 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.

Cyanobacteria, cyanotoxins and lipopolysaccharides in aerosols from inland freshwater bodies and their effects on human bronchial cells.

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.

Microcystin-LR Does Not Alter Cell Survival and Intracellular Signaling in Human Bronchial Epithelial Cells.

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.

Improved multiparametric scrape loading-dye transfer assay for a simultaneous high-throughput analysis of gap junctional intercellular communication, cell density and viability.

Gap junctional intercellular communication (GJIC) is a vital cellular process required for maintenance of tissue homeostasis. In vitro assessment of GJIC represents valuable phenotypic endpoint that could be effectively utilized as an integral component in modern toxicity testing, drug screening or biomedical in vitro research. However, currently available methods for quantifying GJIC with higher-throughputs typically require specialized equipment, proprietary software and/or genetically engineered cell models. To overcome these limitations, we present here an innovative adaptation of traditional, fluorescence microscopy-based scrape loading-dye transfer (SL-DT) assay, which has been optimized to simultaneously evaluate GJIC, cell density and viability. This multiparametric method was demonstrated to be suitable for various multiwell microplate formats, which facilitates an automatized image acquisition. The assay workflow is further assisted by an open source-based software tools for batch image processing, analysis and evaluation of GJIC, cell density and viability. Our results suggest that this approach provides a simple, fast, versatile and cost effective way for in vitro high-throughput assessment of GJIC and other related phenotypic cellular events, which could be included into in vitro screening and assessment of pharmacologically and toxicologically relevant compounds.

Airborne PAHs inhibit gap junctional intercellular communication and activate MAPKs in human bronchial epithelial cell line.

Inhalation exposures to polycyclic aromatic hydrocarbons (PAHs) have been associated with various adverse health effects, including chronic lung diseases and cancer. Using human bronchial epithelial cell line HBE1, we investigated the effects of structurally different PAHs on tissue homeostatic processes, namely gap junctional intercellular communication (GJIC) and MAPKs activity. Rapid (<1 h) and sustained (up to 24 h) inhibition of GJIC was induced by low/middle molecular weight (MW) PAHs, particularly by those with a bay- or bay-like region (1- and 9-methylanthracene, fluoranthene), but also by fluorene and pyrene. In contrast, linear low MW (anthracene, 2-methylanthracene) or higher MW (chrysene) PAHs did not affect GJIC. Fluoranthene, 1- and 9-methylanthracene induced strong and sustained activation of MAPK ERK1/2, whereas MAPK p38 was activated rather nonspecifically by all tested PAHs. Low/middle MW PAHs can disrupt tissue homeostasis in human airway epithelium via structure-dependent nongenotoxic mechanisms, which can contribute to their human health hazards.