Minimum Cytotoxic Concentration Research Articles

Detection of neuroteratogens with an in vitro cytotoxicity assay using primary monolayers cultured from dissociated foetal rat brains

Brain tissues from 19-day-old Sprague-Dawley rat foetuses were trypsinized and then dissociated in a nutrient medium. The resulting cellular suspension was seeded in Falcon tissue-culture flasks and incubated at 37°C in humidified air containing 5% CO 2. The monocell layer obtained after incubation for 3 days consisted of two readily distinguishable cell types: neuronal cells with interconnecting neurites and fascicles, and non-neuronal or glial-type cells that were polygonal in appearance. The cell layers were exposed to graded concentrations of a test chemical dissolved in the nutrient medium. The cultures were examined daily for cell death and for inhibited outgrowth of neurites and fascicles for 3 days and the test was then terminated. Chemicals showing no cytotoxic effect at 2-m m concentration were generally not tested any further. Those found positive were tested at lower concentrations to determine the minimum cytotoxic concentration and the type of cell or cells affected. Of the 109 chemicals examined, 59 were non-cytotoxic at the 2-m m concentration or at the highest concentration at which they were soluble (1 m m for three compounds and 0.5 m for one), ten were preferentially cytocidal for non-neurons, 21 were selectively cytolethal for the neurons and ten were cytocidal for both neurons and non-neurons. The cytotoxicity of the remaining nine chemicals was regarded as unreliable because of the hyperosmolality of their solutions. The neuroteratogenic potential of a chemical, based on cytotoxicity data, was regarded as negligible if the cytocidal effect failed to occur in any cell type at a concentration of ≥2 m m, or if the effect occurred only in non-neurons or in both neurons and non-neurons. The potential was considered positive if cellular degeneration at the lowest cytocidal concentration of a chemical occurred only in the neurons. The agreement between the neuroteratogenic potential in vivo, as reported in the literature, and the cytotoxicity data obtained in this study was very good for 82 chemicals, reasonable for ten, poor for four and undetermined for four. From the results of this investigation and a review of the published teratology data, the neuron and its precursor neuroepithelial cell appear as the most likely initial targets for neuroteratogenic chemicals. Consequently, our in vitro system offers a promising approach for studies on neuroteratogens.