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Organophosphorus (OP) pesticides elicit acute toxicity by inhibiting acetylcholinesterase (AChE), the enzyme responsible for inactivating acetylcholine (ACh) at cholinergic synapses. A number of OP toxicants have also been reported to interact directly with muscarinic receptors, in particular the M 2 muscarinic subtype. Parasympathetic innervation to the heart primarily regulates cardiac function by activating M 2 receptors in the sinus node, atrial-ventricular node and conducting tissues. Thus, OP insecticides can potentially influence cardiac function in a receptor-mediated manner indirectly by inhibiting acetylcholinesterase and directly by binding to muscarinic M 2 receptors. Young animals are generally more sensitive than adults to the acute toxicity of OP insecticides and age related differences in potency of direct binding to muscarinic receptors by some OP toxicants have been reported. We thus compared the effects of the common OP insecticide chlorpyrifos (CPF) on functional signs of toxicity and cardiac ChE activity and muscarinic receptor binding in neonatal and adult rats. Dosages were based on acute lethality (i.e., 0.5 and 1 × LD 10 : neonates, 7.5 and 15 mg/ kg; adults, 68 and 136 mg/kg). Dose-and time-related changes in body weight and cholinergic signs of toxicity (involuntary movements) were noted in both age groups. With 1 × LD 10 , relatively similar maximal reductions in ChE activity (95%) and muscarinic receptor binding (≈ 30%) were noted, but receptor binding reductions appeared earlier in adults and were more prolonged in neonates. In vitro inhibition studies indicated that ChE in neonatal tissues was markedly more sensitive to inhibition by the active metabolite of chlorpyrifos (i.e., chlorpyrifos oxon, CPO) than enzyme in adult tissues (IC 50 values: neonates, 17 nM; adults, 200 nM). Chelation of free calcium with EDTA had relatively little effect on in vitro cholinesterase inhibition, suggesting that differential A-esterase activity was not responsible for the age-related difference in cholinesterase sensitivity between age groups. Pre-incubation of neonatal and adult tissues with selective inhibitors of AChE and butyrylcholinesterase (BChE) indicated that a majority (82-90%) of ChE activity in the heart of both neonates and adults was BChE. The rapid onset (by 4 hours after dosing) of changes in muscarinic receptor binding in adult heart may be a reflection of the more potent direct binding to muscarinic receptors by chlorpyrifos oxon previously reported in adult tissues. The results suggest that ChE activity (primarily BChE) in neonatal heart may be inherently more sensitive to inhibition by some anticholinesterases and that toxicologically significant binding to muscarinic receptors may be

Organophosphorus (OP) insecticides elicit toxicity via acetylcholinesterase inhibition, allowing acetylcholine accumulation and excessive stimulation of cholinergic receptors. Some OP insecticides bind to additional macromolecules including butyrylcholinesterase and cholinergic receptors. While neurotoxicity from OP anticholinesterases has been extensively studied, effects on cardiac function have received less attention. We compared the in vitro sensitivity of acetylcholinesterase, butyrylcholinesterase and [ 3 H]oxotremorine-M binding to muscarinic receptors in the cortex and heart of adult (3 months) and aging (18 months) rats to chlorpyrifos, methyl parathion and their active metabolites chlorpyrifos oxon and methyl paraoxon. Using selective inhibitors, the great majority of cholinesterase in brain was defined as acetylcholinesterase, while butyrylcholinesterase was the major cholinesterase in heart, regardless of age. In the heart, butyrylcholinesterase was markedly more sensitive than acetylcholinesterase to inhibition by chlorpyrifos oxon, and butyrylcholinesterase in tissues from aging rats was more sensitive than enzyme from adults, possibly due to differences in A-esterase mediated detoxification. Relatively similar differences were noted in brain. In contrast, acetylcholinesterase was more sensitive than butyrylcholinesterase to methyl paraoxon in both heart and brain, but no age-related differences were noted. Both oxons displaced [ 3 H]oxotremorine-M binding in heart and brain of both age groups in a concentration-dependent manner. Chlorpyrifos had no effect but methyl parathion was a potent displacer of binding in heart and brain of both age groups. Such OP and age-related differences in interactions with cholinergic macromolecules may be important because of potential for environmental exposures to insecticides as well as the use of anticholinesterases in age-related neurological disorders.

A number of studies have evaluated the possibility that stress-induced changes in blood-brain barrier permeability enhanced the central effects of the carbamate acetylcholinesterase inhibitor, pyridostigmine. We previously found relatively little evidence of stress-induced changes in the acute toxicity of pyridostigmine in rats using a variety of restraint, forced running and forced swimming stress conditions. In this study, we evaluated the effects of sequential pre-exposure to multiple stressors on the acute toxicity of pyridostigmine. Rats (n = 8 per treatment group) were either un-stressed or stressed by restraint (60 min), forced running (60 min, 15 m/min, 6 degrees incline) and forced swimming (15 min), and then given either vehicle (saline, 1 ml/kg, po) or pyridostigmine (30 mg/kg, po) immediately after the final stressor. Functional signs of cholinergic toxicity (involuntary movements, autonomic dysfunction) were recorded at 0.5, 1 and 2 h after dosing. Body temperature was measured both before stress and 2 h after dosing. Rats were sacrificed immediately after 2-h functional observations to collect tissues (whole blood, diaphragm, frontal cortex, hippocampus and cerebellum) for measurement of cholinesterase activity. Stressed rats treated with pyridostigmine exhibited higher lethality (2/8) compared to unstressed rats given pyridostigmine (0/8). Pyridostigmine elicited classical signs of cholinergic toxicity, but the rats that died did not show increased cholinergic signs and no significant differences in cholinergic signs were noted between treatment groups. Cholinesterase activity was significantly inhibited in blood (47-50%) and diaphragm (80%) following pyridostigmine exposure regardless of stress conditions. Slight but significant inhibition (11-15%) of cerebellar cholinesterase activity was observed following pyridostigmine exposure, but inhibition was not influenced by stress. We conclude that while acute lethality from pyridostigmine may be increased by combined, multiple stressors, increased lethality does not appear due to enhanced cholinergic toxicity or via increased cholinesterase inhibition in either central or peripheral tissues.

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