GLUTAMINE-SYNTHETASE ACTIVITY AS A MARKER OF TOXICITY IN CULTURES OF EMBRYONIC CHICK BRAIN AND RETINA CELLS

With the goal of developing a fast and sensitive primary cell culture assay for the determination of neurotoxic potential of compounds, the effect of various toxins on the morphology, cell number (estimated as total cell protein), and glutamine synthetase activity of chick embryo nic neural cells has been tested. Isolated retina or brain cells, grow n as reaggregates in suspension cultures or as monolayers in 24-well p lates, were treated with the substances from day 2 to day 6 after the start of culture. Concentrations causing 50% reduction in protein cont ent in brain cell monolayers were as follows: MeHgCl (0.8 mu M), CdCl2 (1 mu M), 3-acetyl pyridine (0.1 mM), penicillin (above 0.1 mM), diaz epam (0.25 mM), acrylamide (0.3 mM), 2,4,5-T (0.8 mM), lindane (1 mM). In general, retina cells were more sensitive than brain cells. The re aggregate cultures were less sensitive to 1-methyl-4-phenylpyridinium ion (MPP(+)) and cadmium than monolayer cultures, which may be attribu table to their metabolic stability or to diffusional limitations. Glut amine synthetase (GS) activity, measured as glutamate production from glutamine, was a more sensitive indicator of toxicity than total prote in. Retinal cells grown as reaggregates or monolayer cultures, produce d two to four times more glutamate than brain cells grown in a similar fashion. This indicates that retinal glial cell (Muller cell) differe ntiation proceeds in vitro faster than brain astrocyte differentiation , which is consistent with the in vivo developmental pattern. In some cases (methylmercury, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MP P(+), 3-acetyl pyridine, and lindane) a significant increase (as much as 30% of the basal level) was seen in the GS units/mu g cell protein at concentrations of toxins below that reducing total cell protein. Th us, generation of neurotoxic glutamate might play a role in the cell d estruction caused by the chemicals. Other substances (e.g. diazepam an d cadmium) decreased the GS activity considerably, relative to decreas es in total protein. This suggests that these xenobiotics act in a mor e general fashion to reduce metabolic activity and viability.