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Article Excerpt
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.
Key WORDS: apoptosis, differentiation, DNT, human neurospheres, mercury, migration, proliferation. Environ Health Perspect 117:1131-1138 (2009). doi:10.1289/ehp.0800207 available via http://dx.doi.org/ [Online 26 February 2009]
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Developmental neurotoxicity (DNT) of environmental chemicals has been recognized worldwide as a serious threat to human health, and the resulting neurologic deficits negatively affect families and society (Goldman and Koduru 2000; Needleman et al. 2002). Current DNT testing guidelines (Organization for Economic Cooperation and Development 2007; U.S. Environmental Protection Agency 1998) propose investigations in rodents, mainly rats. Such a DNT in vivo testing strategy implies the use of 140 dams and 1,000 pups and is therefore extremely time- and cost-intensive (Lein et al. 2005). Relying solely on the existing guidelines to address current and anticipated future regulatory demands for DNT of the thousands of chemicals for which there are few to no DNT data would incur unacceptable costs in terms of animals and person-years (Lein et al. 2007). Therefore, according to the "3R principle" (reduction, replacement, and refinement) of Russel and Burch (1959), alternative testing strategies are needed to address animal welfare by refining and reducing animal experiments, and to create affordable, sensitive, and mechanism-based methods suitable for high- or medium-throughput screening (Collins et al. 2008). Furthermore, the inclusion of human-cell-based in vitro systems into an integrated DNT tiered testing approach has been recommended to circumvent species differences (Coecke et al. 2007).
To combine transatlantic strengths and avoid doubling of work, a partnership between the Johns Hopkins Center for Alternatives to Animal Testing (Developmental Neurotoxicity TestSmart program), the European Centre for the Validation of Alternative Methods, and the European Chemical Industry Council has been formed. This partnership follows the common goal of "incorporating in vitro alternative methods for DNT testing into international hazard and risk assessment strategies" (Coecke et al. 2007). Coecke et al. (2007) provided a comprehensive overview of the existing in vitro models and stated that, "although all the test systems described were not developed for regulatory purposes at this stage if they prove useful, we hope that this report will encourage their further development to render them amenable to high-throughput approaches."
Therefore, the aim of this work was a) to introduce the cell biological characteristics of human neurospheres as a three-dimensional cell system approach for DNT testing; b) to demonstrate that neurospheres are likely to mirror basic processes of brain development, such as proliferation, differentiation, migration, and apoptosis; and c) to demonstrate that these processes can be modulated by developmental neurotoxicants.
Materials and Methods
Chemicals. We obtained methylmercury chloride (MeHgCl) from Riedel-de Haen (Seelze, Germany); all other substances were obtained from Sigma Aldrich (Munich, Germany), unless otherwise stated.
Cell culture. Cryopreserved normal human neural progenitor cells (hNPCs; Lonza Verviers SPRL, Verviers, Belgium) were cultured at 37[degrees]C and 5% [CO.sub.2] as a suspension culture in proliferation medium consisting of Dulbecco's modified Eagle medium (DMEM) and Hams F12 (3:1) supplemented with B27 (Invitrogen GmbH, Karlsruhe, Germany), 20 ng/mL epidermal growth factor (EGF; Biosource, Karlsruhe, Germany), and 20 ng/mL recombinant human fibroblast growth factor (FGF; R&D Systems, Wiesbaden-Nordenstadt, Germany) (Moors et al. 2007). When spheres reached 0.7 mm in diameter, they were chopped up to passage 3 with a McIlwain tissue chopper. Differentiation was initiated by growth factor withdrawal in differentiation medium [DMEM and Hams F12 (3:1) supplemented with N2 (insulin, transferrin, sodium selenite, putrescine, and progesterone; Invitrogen)] and plated onto poly-D-lysine/laminin-coated chamber slides (BD Bioscience, Erembodegem, Belgium).
Chemical exposure. We exposed cells to indirubin (10 [micro]M) in proliferation medium (28 hr), and to cAMP (200 [micro]M), MeHgCl (250 nM to 1[micro]M), mercury chloride (Hg[Cl.sub.2]; 500 nM to 10 [microu]M, 48 hr) or staurosporine (0.1 and 1 [micro]M), or hydrogen peroxide ([H.sub.2][O.sub.2]; 0.1 and 1 mM) (24...
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