Avian embryonic brain reaggregate culture system. I. Characterization for organophosphorus compound toxicity studies

An avian reaggregate culture system was characterized biochemically and morphologically for use in acute and chronic organophosphorus compound (OP) toxicity studies. Ten-day-old chick embryo brains were dissociated, reaggregated, and maintained in a chemically defined, serum- and antibiotic-free media. Acetylcholinesterase (ACHE), neuropathy target esterase (NTE), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) were examined due to inhibition of these enzymes as a result of acute OP toxicity (ACHE) or delayed toxicity (NTE, CNP). The selected enzymes also indicate reaggregate neuronal (ACHE, possibly NTE), oligodendroglial (CNP), and astrocytic (glutamine synthetase (GS)) activities. Enzyme activities were compared to those in age-matched chick embryo and hatched chick brains. Reaggregate ACHE specific activity was similar to or higher than that of chick embryo or hatched chick. Reaggregate NTE specific activity was initially similar to that of 10-day-old chick embryo, and then increased but subsequently averaged 7.8 nmol/min/mg protein. In chick brain, NTE peaked at hatching and averaged 28 nmol/min/mg protein thereafter. Reaggregate CNP specific activity ranged from 103 to 426 nmol/min/mg protein, whereas activity gradually increased in chick embryo brain to an average of 140 nmol/min/mg protein posthatching. The mean GS activity ranged from 0.15 (Culture Day 4) to 1.09 nmol/min/mg protein (Culture Day 62). Mean protein values per flask ranged from 2.47 to 7.58 mg. Ultrastructurally, myelination was detected at Culture Day 7 and synapses at Day 6. The biochemical and ultrastructural features demonstrate that this reaggregate culture is a practical and sensitive in vitro system for studying both the acute and the long-term neurotoxicological effects of organophosphorus compounds.