Differential effects of triphenylphosphite and diisopropyl phosphorofluoridate on catecholamine secretion from bovine adrenomedullary chromaffin cells.

Types I and II organophosphorus compound-induced delayed neurotoxicity (OPIDN) is characterized by axonal degeneration. Type II compounds, however, uniquely cause cell body damage. Primary cultures of bovine adrenomedullary chromaffin cells were used to investigate and assess biochemically the cell body effects of the Type II compound triphenyl phosphite (TPP). Exocytotic secretion of neurotransmitter was measured to determine whether the cytotoxic action of TPP compromised synaptic events. TPP inhibited catecholamine secretion in both a time- and dose-dependent manner. By 4 h, TPP had inhibited nicotine-induced secretion by about 85%. TPP inhibited catecholamine secretion by about 35% as early as 15 min. The IC50 for TPP was about 45 microM. TPP inhibited secretion regardless of the secretagogue used, although nicotine-induced secretion was inhibited to the greatest extent. The Type I OPIDN diisopropyl phosphorofluoridate (DFP) and the nondelayed-type neurotoxic organophosphorus compound O,O-diethyl-O-4-nitrophenyl phosphate (paraoxon) did not inhibit catecholamine secretion from these cells. In contrast, when high potassium was used to induce secretion, significant stimulation was observed in the presence of DFP and paraoxon. Since Ca2+ homeostasis plays a key role in both exocytosis and neuronal necrosis, its uptake into the cells was measured radiometrically in the presence of TPP or DFP. Incubation with 100 microM TPP for 4 h resulted in the inhibition of 45Ca2+ uptake evoked either by nicotine or K+. No significant inhibition of 45Ca2+ uptake was observed in the presence of DFP. TPP and DFP produced 95% and 88% inhibition, respectively, of the activity of the neurotoxic esterase enzyme (NTE), a putative target for OPIDN. Results suggest that these changes in the secretory mechanisms of the cell may be involved in the TPP-induced pathological alterations in chromaffin cells.

Triphenyl phosphite-induced ultrastructural changes in bovine adrenomedullary chromaffin cells.

Primary cultures of bovine adrenomedullary chromaffin cells were treated with the phosphorus acid ester triphenyl phosphite (TPP), a chemical capable of producing Type II organophosphorus compound-induced delayed neurotoxicity (OPIDN), and the morphological changes were assessed by transmission electron and scanning microscopy. Following a 24-hr incubation with 100 microM TPP nearly all mitochondria were either disrupted or swollen and glycogen buildup within the cytoplasm was evident. The viability of cells treated with TPP and cultured on coverslips for scanning electron microscopy was very low. By scanning electron microscopy, the filopodia of these cells appeared contracted. The surface texture was very irregular and giant globular bodies were evident. Parallel studies were carried out with the cholinergic compound O,O-diethyl 4-nitrophenyl phosphate (paraoxon) and the Type I delayed neurotoxicant O,O-diisopropylphosphorofluoridate (DFP). Transmission and scanning electron microscopy revealed that treatment with these organophosphorus compounds did not produce the ultrastructural effects that were seen with TPP. The morphological data were confirmed biochemically by assessing the viability of the mitochondria via measurement of [3H]adenosine incorporation into ATP. Treatment with 100 microM TPP for 4 or 24 hr caused a marked inhibition (90% relative to controls) of adenosine incorporation. Neither 100 microM paraoxon nor 100 microM DFP had an inhibitory effect on incorporation. The effect of TPP was time-dependent with significant biochemical effects as early as 60 min. In contrast, ultrastructural changes were not seen until 24 hr. Morphologically, the 60-min incubations showed no perturbation in mitochondrial integrity. Our results support a specific effect of the triphenylphosphite, TPP, a Type II OPIDN compound, not a general toxic effect of organophosphorus compounds since the cholinergic agent paraoxon and the Type I delayed neurotoxic compound DFP did nto alter the cells ultrastructurally or compromise the mitochondria biochemically. The apparent target for TPP toxicity is the mitochondria.