An assay for neurotoxic esterase (neuropathy target esterase, NTE) was developed by Johnson (1,2) to assess the delayed neurotoxic potential of organophosphorus compounds. NTE activity is calculated from the rate of phenyl valerate hydrolysis resistant to paraoxon and sensitive to mipafox inhibition under specified conditions of inhibitor concentrations, pH, temperature, and incubation times with inhibitors and substrate. The amount of phenol produced is measured colorimetrically after its oxidative coupling with 4-aminoantipyrine to yield 4-N-(1,4-benzoquinoneimine)-antipyrine, a chromophore with a wavelength of maximum absorbance (lambda m) 510 nm and corresponding molar absorptivity (molar extinction coefficient, epsilon) equal to 13,900 M-1cm-1. The assay was improved and simplified later by Johnson (3) without any change in the lambda m or epsilon, even though the chromophore solvent was altered by adding the detergent, sodium dodecyl sulfate (SDS). The present work demonstrates that when the NTE assay is performed according to the improved procedure, with a final [SDS] of 3.0 mg/mL, the lambda m of the chromophore in the assay mixture is shifted from 510 to 490 nm. The same shift in the chromophore lambda m is observed when phenol standards are coupled with 4-aminoantipyrine in solutions containing 3.0 mg/mL SDS. A systematic investigation of the dependence of the lambda m of the chromophore on [SDS] in the assay mixture revealed that the spectral shift increases rapidly at an [SDS] greater than the apparent critical micelle concentration (CMC; estimated to be 0.53 mg/mL under these conditions) and begins to plateau at [SDS] greater than 10 mg/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorpyrifos (CPS; O,O-diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate; Dursban) is a widely used broad-spectrum organophosphorus (OP) insecticide. Because some OP compounds can cause a sensory-motor distal axonopathy called OP compound-induced delayed neurotoxicity (OPIDN), CPS has been evaluated for this paralytic effect. Early studies of the neurotoxicity of CPS in young and adult hens reported reversible leg weakness but failed to detect OPIDN. More recently, a human case of mild OPIDN was reported to result from ingestion of a massive dose (about 300 mg/kg) in a suicide attempt. Subsequent experiments in adult hens (the currently accepted animal model of choice for studies of OPIDN) showed that doses of CPS in excess of the LD50 in atropine-treated animals inhibited brain neurotoxic esterase (NTE) and produced mild to moderate ataxia. Considering the extensive use of CPS and its demonstrated potential for causing OPIDN at supralethal doses, additional data are needed to enable quantitative estimates to be made of the neuropathic risk of this compound. Previous work has shown that the ability of OP insecticides to cause acute cholinergic toxicity versus OPIDN can be predicted from their relative tendency to inhibit the intended target, acetylcholinesterase (AChE), versus the putative neuropathic target, NTE, in brain tissue. The present study was designed to clarify the magnitude of neuropathic risk associated with CPS exposures by measuring hen brain AChE and NTE inhibition following dosing in vivo and determining the bimolecular rate constant of inhibition (ki) for each enzyme by the active metabolite, CPS oxon (CPO), in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Xanthine oxidase (XO) has been proposed as an important source of free radicals during ischemia. This enzyme normally exists as a dehydrogenase (XD), but it is converted to XO in some ischemic tissues. Recently, treatment of animals with the XD and XO inhibitor allopurinol or with free radical scavengers before cerebral ischemia has been shown to reduce brain injury. Therefore, we studied conversion of XD to XO in three ischemic and nonischemic brain regions during focal cerebral ischemia resulting from permanent occlusion of the middle cerebral artery (MCAO) in anesthetized rats. In nonischemic brain, 16-22% of the enzyme was in the XO form. After 24 h of ischemia this value was not significantly different (10-15%). Neither the total activity of XO nor that of XD changed, indicating that there was no irreversible conversion of XD to XO. To further explore the possible role of XO, we examined the effect of various doses of allopurinol (5, 20, or 100 mg/kg given 1 h before MCAO or 100 mg/kg given 48, 24, and 1 h before MCAO) on uric acid accumulation, brain edema formation, and cerebral blood flow (CBF) 24 h after MCAO. All but the lowest dose of allopurinol greatly reduced the appearance of uric acid in the ischemic brain; however, only the highest dose of allopurinol had any beneficial effect on brain edema. This reduction in brain edema occurred without a significant improvement in CBF. Thus XO is probably not an important source of free radicals in this model of focal cerebral ischemia.

Previous work has shown that acute exposures to chlorpyrifos (CPS; diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate) cannot produce > 70% inhibition of brain neurotoxic esterase (NTE) and cause organophosphorus compound-induced delayed neurotoxicity (OPIDN) unless the dose is well in excess of the LD50, necessitating aggressive therapy for cholinergic toxicity. The present study was carried out to determine if repeated doses of CPS at the maximum tolerated daily dose without prophylaxis against cholinergic toxicity could cause cumulative inhibition of NTE and OPIDN. Adult hens were dosed daily for 20 days with CPS (10 mg/kg/day po in 2 ml/kg corn oil) or corn oil (vehicle control) (2 ml/kg/day po) and observed for an additional 4 weeks. Brain acetylcholinesterase (AChE), brain and lymphocyte NTE, and plasma butyrylcholinesterase (BuChE) activities were assayed on Days 0 (control only), 4, 10, 15, 20, and 48. During Days 4-20, brain AChE and plasma BuChE activities from CPS-treated hens were inhibited 58-70% and 49-80% of contemporaneous controls, respectively. At 4 weeks after the end of dosing, brain AChE activity in treated birds had recovered to 86% of control and plasma BuChE activity was 134% of control. Brain and lymphocyte NTE activities of treated animals throughout the study were 82-99% and 85-128% of control, respectively. Neither brain nor lymphocyte NTE activities in treated hens exhibited cumulative inhibition. The 18% inhibition of brain NTE seen on days 10 and 20 was significant, but substantially below the putative threshold for OPIDN.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical manifestations of mild organophosphorus compound-induced delayed neurotoxicity (OPIDN) produced by diisopropylphosphorofluoridate (DFP) in adult hens are potentiated by posttreatment with phenylmethanesulfonyl fluoride (PMSF). The purpose of this study was to assess whether potentiation of mild OPIDN produces a pattern of axonal lesions in the central and peripheral nervous system similar to that seen in severe OPIDN. Groups of 6 hens each were given the following priming/challenge doses sc at 0 and 4 h, respectively: 0.20 ml/kg corn oil/0.50 ml/kg glycerol formal (GF) (control); 0.50 mg/kg DFP/GF (low-dose DFP); 0.50 mg/kg DFP/60 mg/kg PMSF (potentiated DFP); 60 mg/kg PMSF/GF (PMSF alone); 60 mg/kg PMSF/1.5 mg/kg DFP (protected DFP); and 1.5 mg/kg DFP/GF (high-dose DFP). Two hens from each group were used to assay brain neurotoxic esterase (NTE) 24 h after the challenge dose, and the remaining hens were scored for deficits in walking, standing, and perching ability on d 18. Three hens from each group were perfusion-fixed on d 22 and neural tissues were prepared for histologic evaluation. DFP and/or PMSF caused > 88% brain NTE inhibition in all treated groups, compared to control. Protected DFP yielded no clinical deficits and a distribution and frequency of axonal lesions similar to control. PMSF alone produced a small increase in the frequency of lesions in the cervical spinal cord and peripheral nerves compared to control. Low-dose DFP caused minimal ataxia and increased frequency of axonal lesions in dorsal and lateral cervical spinal cord, ventral lumbar spinal cord, and inferior cerebellar peduncles (ICP) compared to control. Potentiated DFP and high-dose DFP produced maximal ataxia and essentially identical increases in the frequency of lesions in dorsal and ventral thoracic spinal cord, lateral lumbar spinal cord, and peripheral nerves compared to low-dose DFP. The results indicate that PMSF potentiation of mild OPIDN induced in adult hens by low-dose DFP results in an overall pattern of axonal degeneration like that produced by a threefold higher dose of DFP alone, and support the hypothesis that potentiation causes an increase in the frequency of axonal lesions in central and peripheral loci normally affected by OPIDN.