Human neurospheres as three-dimensional cellular systems for developmental neurotoxicity testing.

BACKGROUND: Developmental neurotoxicity (DNT) of environmental chemicals is a serious threat to human health. Current DNT testing guidelines propose investigations in rodents, which require large numbers of animals. With regard to the "3 Rs" (reduction, replacement, and refinement) of animal testing and the European regulation of chemicals [Registration, Evaluation, and Authorisation of Chemicals (REACH)], alternative testing strategies are needed in order to refine and reduce animal experiments and allow faster and less expensive screening. OBJECTIVES: The goal of this study was to establish a three-dimensional test system for DNT screening based on human fetal brain cells. METHODS: We established assays suitable for detecting disturbances in basic processes of brain development by employing human neural progenitor cells (hNPCs), which grow as neurospheres. Furthermore, we assessed effects of mercury and oxidative stress on these cells. RESULTS: We found that human neurospheres imitate proliferation, differentiation, and migration in vitro. Exposure to the proapoptotic agent staurosporine further suggests that human neurospheres possess functioning apoptosis machinery. The developmental neurotoxicants methylmercury chloride and mercury chloride decreased migration distance and number of neuronal-like cells in differentiated hNPCs. Furthermore, hNPCs undergo caspase-independent apoptosis when exposed toward high amounts of oxidative stress. CONCLUSIONS: Human neurospheres are likely to imitate basic processes of brain development, and these processes can be modulated by developmental neurotoxicants. Thus, this three-dimensional cell system is a promising approach for DNT testing.

Lightening up the UV response by identification of the arylhydrocarbon receptor as a cytoplasmatic target for ultraviolet B radiation.

UVB radiation-induced signaling in mammalian cells involves two major pathways: one that is initiated through the generation of DNA photoproducts in the nucleus and a second one that occurs independently of DNA damage and is characterized by cell surface receptor activation. The chromophore for the latter one has been unknown. Here, we report that the UVB response involves tryptophan as a chromophore. We show that through the intracellular generation of photoproducts, such as the arylhydrocarbon receptor (AhR) ligand 6-formylindolo[3,2-b]carbazole, signaling events are initiated, which are transferred to the nucleus and the cell membrane via activation of the cytoplasmatic AhR. Specifically, AhR activation by UVB leads to (i) transcriptional induction of cytochrome P450 1A1 and (ii) EGF receptor internalization with activation of the EGF receptor downstream target ERK1/2 and subsequent induction of cyclooxygenase-2. The role of the AhR in the UVB stress response was confirmed in vivo by studies employing AhR KO mice.

ERK-dependent and -independent pathways trigger human neural progenitor cell migration.

Besides differentiation and apoptosis, cell migration is a basic process in brain development in which neural cells migrate several centimeters within the developing brain before reaching their proper positions and forming the right connections. For identifying signaling events that control neural migration and are therefore potential targets of chemicals to disturb normal brain development, we developed a human neurosphere-based migration assay based on normal human neural progenitor (NHNP) cells, in which the distance is measured that cells wander over time. Applying this assay, we investigated the role of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the regulation of NHNP cell migration. Exposure to model substances like ethanol or phorbol 12-myristate 13-acetate (PMA) revealed a correlation between ERK1/2 activation and cell migration. The participation of phospho-(P-) ERK1/2 was confirmed by exposure of the cells to the MEK inhibitor PD98059, which directly prohibits ERK1/2 phosphorylation and inhibited cell migration. We identified protein kinase C (PKC) and epidermal growth factor receptor (EGFR) as upstream signaling kinases governing ERK1/2 activation, thereby controlling NHNP cell migration. Additionally, treatments with src kinase inhibitors led to a diminished cell migration without affecting ERK1/2 phosphorylation. Based on these results, we postulate that migration of NHNP cells is controlled via ERK1/2-dependent and -independent pathways.

Polychlorinated biphenyls disturb differentiation of normal human neural progenitor cells: clue for involvement of thyroid hormone receptors.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental chemicals that accumulate in adipose tissues over the food chain. Epidemiologic studies have indicated that PCBs influence brain development. Children who are exposed to PCBs during development suffer from neuropsychologic deficits such as a lower full-scale IQ (intelligence quotient), reduced visual recognition memory, and attention and motor deficits. The mechanisms leading to these effects are not fully understood. It has been speculated that PCBs may affect brain development by interfering with thyroid hormone (TH) signaling. Because most of the data are from animal studies, we established a model using primary normal human neural progenitor (NHNP) cells to determine if PCBs interfere with TH-dependent neural differentiation. NHNP cells differentiate into neurons, astrocytes, and oligodendrocytes in culture, and they express a variety of drug metabolism enzymes and nuclear receptors. Like triiodothyronine (T3), treatment with the mono-ortho-substituted PCB-118 (2,3',4,4 ,5-pentachlorobiphenyl; 0.01-1 microM) leads to a dose-dependent increase of oligodendrocyte formation. This effect was congener specific, because the coplanar PCB-126 (3,3',4,4 ,5-pentachlorobiphenyl) had no effect. Similar to the T3 response, the PCB-mediated effect on oligodendrocyte formation was blocked by retinoic acid and the thyroid hormone receptor antagonist NH-3. These results suggest that PCB-118 mimics T3 action via the TH pathway.