Chick embryo neural retinal cell culture as a screen for developmental toxicity

Abstract

An in vitro screen for developmental toxic potential of chemicals using primary cultures of chick embryo neural retina cells in described. The neural retinas of incubation Day 6.5 White Leghorn chick embryos are dissociated into single cells, which are subsequently maintained in a rotating suspension culture. Under normal circumstances, neural retina cells form spheroidal aggregates of a consistent size over the first 24 hr of culture, an event which is dependent on competent cell-cell interactions. Over the remaining 7-day period of culture, cells continue to divide and grow, and differentiation takes place. Each of these developmentally important events—aggregation, growth, and differentiation—is objectively and quantitatively measured as aggregate size and number, aggregate protein content, and glutamine synthetase (a marker of differentiation) activity, respectively. The effects on each developmental endpoint of 22 chemicals, 14 of which have been demonstrated to be developmentally toxic in one or more mammalian species in vivo, and 8 of which are not developmentally toxic, were evaluated. Chemicals were tested up to a concentration of 40 m m, or until marked cytolethality was observed. Of the known developmental toxicants, all but one, 2-methoxyethanol, affected one or more endpoints in the assay. The teratogenic metabolite of 2-methoxyethanol, 2-methoxyacetic acid, was active in the assay. None of the 8 nondevelopmental toxicants had any effect up to c concentration of 40 m m, or at biologically achievable concentrations (e.g., in vivo systemic concentrations at the LD50). Thus, the assay is 95% concordant with in vivo results for this set of chemicals. Quantitative comparisons were made (1) between developmentally toxic ip dosages in rats or mice in vivo and effective concentrations in the chick retina cell culture, and (2) between effective concentrations in chick retina cell culture and rodent whole embryo culture. In the first instance, 71% of the comparisons, and in the second instance, 89% of the comparisons, were within the same order of magnitude (and usually within a factor of two), indicating that the chick retina cell culture is also concordant with developmental toxic potency. Last, it was observed that test agents differentially affect developmental endpoints. Because the assay's endpoints are measured separately and objectively, it may be possible to use the assay to evaluate the effects of test agents on cellular development. The effects of tested chemicals on chick neural retina cells was consistent with reported effects of those chemicals. For example, chemicals which interfere with intercellular communication affected aggregation (e.g., all-trans retinoic acid, TPA), heavy metals (CdCl 2, HgCl 2) diminished growth, and chemicals which affect gene expression inhibited differentiation (e.g., bromodeoxyuridine, actinomycin D). Thus, the chick embryo retina assay appears to be capable of recapitulating in vivo developmental toxicity, and also may be useful in providing information about the mode of action of a developmental toxicant at the cellular level.