Joseph H. Fleisher

Dealkylation as a mechanism for aging of cholinesterase after poisoning with pinacolyl methylphosphonofluoridate

Abstract The decrease in the mole fraction of 32 P-pinacolyl methylphosphonic acid ( 32 P-PMPA) derived from acetylcholinesterase inhibited with 32 P-labeled pinacolyl methylphosphonofluoridate (Soman) was used to measure the rate of dealkylation of the phosphonylated enzyme in vitro . First-order rate constants for dealkylation and for “aging,” as measured by decrease in reactivatability by monoisonitrosoacetone were not significantly different. Dealkylation in the brain of the rat poisoned with 32 P-Soman in vivo reached a maximum within 30 min after poisoning. Eserine, given prior to poisoning with 32 P-Soman in vivo , raised the LD 50 of Soman 3–8-fold and appeared to shunt the Soman to nondealkylating sites, as judged by the high mole-fraction of 32 P-PMPA and the large retention of cholinesterase activity in the brains of these animals.

Antagonism of sarin poisoning in rats and guinea pigs by atropine, oximes, and mecamylamine

Abstract Intramuscular atropine-oxime therapy for sarin poisoning in rats and guinea pigs was evaluated. Injection of 7.5 mg/kg of pyridinium aldoxime methochloride (2-PAMCl) plus 16 mg/kg of atropine 1 min after poisoning rats with sarin subcutaneously raised the LD50 of the organophosphorous compound 2.5-fold. Toxogonin (7.5 mg/kg) gave somewhat less protection when substituted for 2-PAMCl. Atropine-oxime therapy was more successful in sarin-poisoned guinea pigs, the LD50 being increased 18.5- and 13.6-fold by 7.5 mg/kg of 2-PAMCl and Toxogonin, respectively. Addition of 1 mg/kg of mecamylamine to therapy with atropine-oximes gave added protection to sarin-poisoned rats. Increase of Toxogonin dosage to 17.0 mg/kg, the molar equivalent of 7.5 mg/kg of 2-PAMCl, significantly increased the protection offered by atropine-oxime treatment to both sarin-poisoned rats and guinea pigs. A dose of 30 mg/kg of 2-PAMCl given orally 1 hour prior to poisoning with sarin and injection of atropine im 1 min after poisoning appeared to be ineffective in rats but raised the LD50 of sarin 6.3-fold in guinea pigs.

Dealkylation and loss of capacity for reactivation of cholinesterase inhibited by sarin.

Inhibition of rat brain acetylcholinesterase by 32P-sarin in vivo results initially in 32P-isopropylmethylphosphonylated enzyme. The percentage of inhibited enzyme that could not be reactivated by pyridinium aldoxime methochloride (aged enzyme) approximated the amount of radioactivity identified as 32P-methylphosphonate. The 32P-isopropyl methylphosphonate not released fromthe inhibited enzyme by the oxime accounted for 51 � 10 percent ( standard deviation) of the radioactivity fixed to brain tissue. It showed no correlation with aging and was probably bound to sites other than acetylcholinesterase.

The effects of atropine-oxime therapy on cholinesterase activity and the survival of animals poisoned with O,O-diethyl-O-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate

Abstract Brain and diaphragm cholinesterase (ChE) activity in rats was reduced to 22.6 ± 4.3 and 15.5 ± 6.3% ( P = 0.95) of normal respectively 24 hours after oral dosage with 235 mg/kg (0.8 LD50) of O,O -diethyl- O -(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate (Diazinon). The ChE activity of brain and diaphragm in poisoned rats recovered gradually to 44.8 ± 8.9 and 62.6 ± 7.5% ( P = 0.95) of normal, respectively 140 hours after poisoning. Treatment with 16 mg/kg of atropine im 10 minutes after poisoning followed 24 hours later by 30 mg/kg of pyridine 2-aldoxime methochloride (2-PAM Cl) by either the oral or iv route resulted in significant reactivation of diaphragm ChE levels compared to poisoned controls. The oral single LD50 of Diazinon in rats treated with 16 mg/kg of atropine im along with 30 mg/kg of 2-PAM Cl orally or iv was elevated 3.1 and 1.7 times, respectively. Administration of 2-PAM Cl to Diazinon-poisoned rabbits resulted in reactivation of inhibited blood ChE activity concurrent with decrease in signs of poisoning. Within 2 hours the animals were again weak and ataxic, and blood ChE showed renewed inhibition. These observations suggest that effective therapy of Diazinon intoxication requires the maintenance of a level of oxime high enough to reactivate inhibited ChE during the time in which active inhibitor is formed or available. This may be more readily achieved when 2-PAM Cl is given orally following an initial iv dose in conjunction with atropine given im.

Effects of 2-pyridinium aldoxine methochloride and atropine in relation to elevation of blood pH in soman-poisoned dogs

Abstract The effects of various chemical treatments on the respiratory and cardiovascular deficits caused by poisoning dogs with pinacolyl methylphosphonofluoridate (soman) and on survival of the dogs were studied. A comparison was made between treatment of the poisoned dogs with atropine alone and with atropine plus 2-pyridinium aldoxime methochloride (PAM). Intravenous injection of 1 mg/kg of atropine and 104 mg/kg of PAM within 2 min after poisoning dogs with 30 μg/kg of soman i.v. (approximately 3 times the ld50) counteracted the respiratory and cardiovascular collapse produced by soman and saved 12 of 13 animals. Phenoxybenzamine, 0.5 mg/kg, injected i.v. immediately after the onset of hypertension resulting from treatment of soman-poisoned dogs with atropine and PAM affected a more rapid return to normal blood pressure levels without impairing survival. The half-time for loss of reactivatability of soman-inhibited red blood cell acetylcholinesterase (RBC (AChE) in vivo was 5.55 min at pH 7.4 and 12.50 min at pH 7.8 (achieved by pretreatment of the dogs with Tris buffer. At the higher pH, administration of PAM could be delayed for 20 min after poisoning with 50 μg/kg of soman, and 23.4 per cent of the inhibited RBC ChE was reactivated and 80 per cent of the dogs were saved. When the blood was at physiological pH, dogs poisoned with soman and given PAM 20 min later showed little or no recovery of inhibited RBC AChE and complete mortality.

Dephosphorylation in vivo of brain acetylcholinesterase inhibited by isopropyl methylphosphonofluoridate (sarin)

Abstract Intravenous injection of32P-isopropyl methylphosphonofluoridate (Sarin) into rats resulted in inhibition of brain acetylcholinesterase (AChE) activity and phosphonylation of the enzyme. Spontaneous recovery from inhibition occurred between 0.5 and 48 hr in the Sarin-intoxicated animals in correlation with comparable dephosphorylation of Sarin-derived phosphorus bound to the enzyme. Injection of 44 mg/kg of monoisonitrosoacetone (MINA) into the Sarin-intoxicated rats resulted in significant reactivation of inhibited brain AChE over and above that occurring spontaneously, accompanied by additional dephosphorylation of enzymatically bound phosphorus. Loss of phosphorus in vivo took place exclusively as 32P-isopropyl methylphosphonic acid (32P-IMPA). No significant loss of Sarin-derived32P bound to AChE as 32P-methylphosphonate was apparent under these conditions.

Reactivatability of soman-inhibited end plate cholinesterase and neuromuscular transmission

Abstract The effect of high doses of 2-pyridine aldoxime methochloride (2-PAMC1) on neuromuscular blockade in anesthetized, artificially respired, atropinized dogs poisoned with pinacolyl methylphosphonofluoridate (soman) was studied. A comparison was made between return of neuromuscular function following early administration of 2-PAMCl PAMC1 and that following late administration of the oxime. Studies were also made of recovery of neuromuscular function in soman-poisoned dogs in the absence of the oxime. By means of a histochemical technique, effects of soman and 2-PAMC1 on neuromuscular function were correlated with end plate acetylcholinesterase (AChE) activity. It was found that prompt intravenous injection of 104 mg/kg of 2-PAMC1 following 10 × LD50 dose of soman iv rapidly reverses neuromuscular blockade and restores measurable endplate AChE. The oxime was ineffective when given later than 30 min after the administration of soman. The reactivating effect of 2-PAMC1 in the dog only occurred when the dose was in excess of 50% of the LD50 of the oxime. Partial recovery of neuromuscular transmission occurred in the absence of detectable AChE. “Aging” of rat diaphragm end plate AChE inhibited by soman in vitro also was investigated. End plate AChE lost its capacity to be reactivated by 2-PAMC1 within 2 hours after incubation with soman. Preincubation with isopropyl methylphosphonofluoridate (sarin) before soman enabled the oxime to reactivate the inhibited enzyme.