MODELING THE DEVELOPMENTAL NEUROTOXICITY OF CHLORPYRIFOS IN-VITRO - MACROMOLECULE SYNTHESIS IN PC12 CELLS

Exposure to apparently subtoxic doses of chlorpyrifos during late stag es of brain development affects cell acquisition through a mixture of cholinergic and noncholinergic mechanisms. In the current study, we mo deled these effects in vitro using rat pheochromocytoma (PC12), a cell line that, upon nerve-growth factor (NGF)-induced differentiation, de velops the appearance and function of cholinergic target neurons, incl uding the expression of cholinergic receptors. In the undifferentiated state (no NGF), chlorpyrifos evoked an immediate (1 h), robust, conce ntration-dependent inhibition of DNA synthesis as evaluated by [H-3]th ymidine incorporation, with a threshold of 0.5-1.5 mu g/ml. Continuous exposure for up to 24 h maintained the same degree of inhibition. The effects were selective for DNA synthesis, as much smaller inhibitions were found for synthesis of RNA or protein. In contrast, direct choli nergic stimulation of the cells by 100 mu M nicotine had much smaller effects on DNA synthesis. Moreover, the effects of chlorpyrifos on DNA synthesis could not be blocked by nicotinic or muscarinic antagonists , confirming that the effects were not mediated primarily through chol inergic hyperstimulation consequent to cholinesterase inhibition or to direct receptor-mediated effects. When PC12 cells underwent NGF-induc ed differentiation, the rate of cell replication fell dramatically and neurite extension was evident both from morphological examination and from biochemical markers (increased protein:DNA ratio). After introdu ction of NGF, chlorpyrifos maintained its ability to inhibit DNA synth esis acutely. However, the ability to inhibit RNA and protein synthesi s initially intensified and then disappeared, indicating a shift in ma cromolecular targets as differentiation proceeded. We also tested the effects of long-term exposure to chlorpyrifos during the process of NG F-induced differentiation. Continuous chlorpyrifos exposure resulted i n severe reductions in macromolecule synthesis and a deficit in the to tal number of cells, effects similar to those seen with chlorpyrifos t reatment in vivo. At the highest concentrations, neurite extension was also inhibited. Our results suggest that chlorpyrifos can interact di rectly with developing neural cells to inhibit replication and neuriti c outgrowth. (C) 1998 Academic Press.