Development mouse neuroblastoma cells

The cultured cells that we have been studying are immature and may therefore be models for the developing brain rather than the mature brain. The mouse neuroblastoma cells, however, can be induced to differentiate by exposure to 0.5mM sodium butyrate (Fig 7). This causes an increase in cytoplasm nucleus ratio, neurite outgrowth and induction of tyrosine hydroxylase and other enzymes. As shown in Fig 8, butyrate caused an increase in T3 uptake at 2 hours. The initial (l min) uptake was also increased. The apparent Ka of nuclear binding in intact cells with the finding that the apparent affinity of the nuclear receptor was decreased by butyrate but the Ka in isolated nuclei was not affected. The apparent inconsistency of an increased intracellular transport but a decreased intracellular free T/ concentration is unexplained. As described by others in developing rats and in cultured rat glial cells we also found that neuroblast differentiation was accompanied by an increase in nuclear receptor number. 

Other tissue culture studies of MeHg effects have utilized a variety of cell types, including mouse leukemic cells, mouse glioma cells, HeLa S3 cells, and mouse neuroblastoma cells. "" Although there ai certain merits to using neoplastic cells for the study of MeHg toxicity, the relevance of these models to the effects of MeHg upon fetal brain development is open to question. 

The implementation of animal test protocols in the 1980s has been accompanied by the development of a host of alternative methods to study adverse effects of chemicals on reproductive and developmental parameters. For example, rat whole embryo culture stems from the seventies (16), as does the rat limb bud organ culture (17) and rat limb bud and brain micromass was developed in the eighties (18). An elegant nonvertebrate alternative model used regeneration of polyps of Hydra atUnuata from dissociated cells (19). Animal-free in vitro alternatives include those employing the proliferation of a human embryonic palatal mesenchymal cell line (20), the attachment of a mouse ovarian tumor cell line (21), and the differentiation of a neuroblastoma cell line (22) and a embryonal carcinoma cell line (23). Various overviews of methods have been published over the years (24). The predictability of... 

Other biological assays have been developed that avoid the sacrifice of animals. The mouse neuroblastoma assay measures PSTs by the survival of cultured neuroblastoma cells after addition of extract. In this assay, Na /K -ATPase inhibitor ouabain and the sodium channel activator verattidine are added to neuroblastoma cells prior to the addition of toxin extract. PSTs that are present in the extract prevent the veratridine-induced influx of sodium ions into the cells, and thus prevent cell death. A commercially available kit version of this assay, the MIST shippable cell bioassay kit [184], showed good agreement in a comparative study to the mouse bioassay [185] however, it performed unsatisfactorily in an AOAC international collaborative study in 1999, and there have been quality problems related to the shipping of the kit. The MIST kit was eventually replaced by the MIST Alert kit, which is an immunological assay that utilizes antibodies to STX, neoSTX, GTXl-4 [186]. Various other immunoassays exist [187]. 

The simplest in vitro model, the cell line, is a population of cells that can be maintained in culture for an extended period of time. Neuronal cell lines normally originate from a single common ancestor cell (clonal) and are often derived from tumors, e.g., pha-eochromocytomas (adrenal medullary tumor) (e.g., PC12 cell line) [13] and neuroblastomas [14] such as mouse N2a or human SH-SY5Y. However, a recently developed technique to introduce oncogenes into primary cultures through retroviruses has opened up new possibilities [15]. 

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