Inhibition Of Brain Neurotoxic Esterase Research Articles

POTENTIATION OF ORGANOPHOSPHORUS COMPOUND-INDUCED DELAYED NEUROTOXICITY (OPIDN) IN THE CENTRAL AND PERIPHERAL NERVOUS SYSTEM OF THE ADULT HEN: DISTRIBUTION OF AXONAL LESIONS

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. Croups 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 (CF) (control); 0.50 mg/kg DFP/CF (low-dose DFP); 0.50 mg/kg DFP/60 mg/kg PMSF (potentiated DFP); 60 mg/kg PMSF/CF (PMSF alone); 60 mg/kg PMSFII.5 mg/kg DFP (protected DFP); and 1.5 mg/kg DFP/CF (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 perfu-sion-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.

Chlorpyrifos: Assessment of Potential for Delayed Neurotoxicity by Repeated Dosing in Adult Hens with Monitoring of Brain Acetylcholinesterase, Brain and Lymphocyte Neurotoxic Esterase, and Plasma Butyrylcholinesterase Activities

Chlorpyrifos: Assessment of Potential for Delayed Neurotoxicity by Repeated Dosing in Adult Hens with Monitoring of Brain Acetylcholinesterase, Brain and Lymphocyte Neurotoxic Esterase, and Plasma Butyrylcholinesterase Activities. Richardson, R. J., Moore, T. B., Kayyali, U. S., and Randall, J. C. (1993). Fundam. Appl. Toxicol. 21, 89-96.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 significantly, but substantially below the putative threshold for OPIDN. Body weight of treated hens decreased 10-25% during Days 4-20 and recovered to 87% of control by the end of the study. Some treated hens developed a slight staggering gait during the first week of dosing, which disappeared by the second week. Throughout the 4-week observation period, all hens appeared normal and were able to perch on a horizontal rod. The results indicate that daily dosing with CPS at a level sufficient to cause significant loss of body weight as well as marked inhibition of brain AChE and plasma BuChE resulted in no significant change in lymphocyte NTE activity, a maximum inhibition of brain NTE of 18%, no cumulative inhibition of lymphocyte or brain NTE, and no clinical signs of OPIDN.

Murine susceptibility to organophosphorus-induced delayed neuropathy (OPIDN)

This study reports that CD-1 strain mice are neuropathologically and biochemically responsive to acute doses of tri-ortho-cresyl phosphate (TOCP). Young (25–30 g) male and female animals were exposed (po) to a single dose of TOCP (580–3480 mg/kg) and sampled for neurotoxic esterase (NTE) activity at 24 and 44 hr postexposure and for neuropathic damage 14 days later. Biochemically, high intragroup variability existed at the lower doses, and at higher levels of TOCP exposure (i.e., ≥ 1160 mg/kg), mean brain NTE inhibition never exceeded 68%. Hen and mouse brain NTE activity, assayed in vitro for sensitivity to inhibition by tolyl saligenin phosphate (TSP), the active neurotoxic metabolite of TOCP, showed similar IC50 values. Histologically, highly variable spinal cord damage was recorded throughout treatment groups and mean damage scores followed a dose-response pattern with no apparent correlation to threshold (i.e., ≥ 65%) inhibition of brain NTE activity. Topographically, axonal degeneration in the mouse spinal cord predominated in the lateral and ventral columns of the upper cervical cord. Unlike the rat, which displays degeneration in the upper cervical cord's dorsal columns (i.e., gracilis fasciculus) in response to TOCP intoxication, treated mice showed minimal damage to this tract. To examine this discrepancy further, ultrastructural morphometric analysis of axon diameters in the cervical cord was performed in control mice and rats. These results indicated that in both species, the largest diameter (≥ 4 μm) axons are housed in the ventral columns of the cervical spinal cord, suggesting that axon length and diameter may not be the only criteria underlying fiber tract vulnerability in OPIDN.

Assessment of the Delayed Neurotoxicity of Tributyl Phosphate, Tributoxyethyl Phosphate, and Dibutylphenyl Phosphate

There industrial organophosphorus compounds were tested for their ability to cause organophosphorus compound-induced delayed neurotoxicity (OPIDN) in the adult hen. The compounds tested were tributyl phosphate (TBP), tributoxyethyl phosphate (TBEP), and dibutylphenyl phosphate (DBPP). The acute oral LD50 of TBP and DBPP were estimated to be 1,863 and 1,500 mg/kg, respectively, and the dose equal to the LD50 was used as a test dose. The acute oral LD50 of TBEP was greater than 5,000 mg/kg and 5,000 mg/kg was used as a test dose. An oral dose of 750 mg tri-o-cresyl phosphate (TOCP) was used as a positive control. For the acute delayed neurotoxicity test, hens were given two test doses of the test materials 21 days apart and killed 21 days after the second dose. None of the hens given TBP, TBEP, or DBPP exhibited nerve damage or clinical signs which distinguished them from untreated control animals. A single dose of TOCP resulted in paralysis and a histopathological profile typical of a distal neuropathy. For the assay of the inhibition of esterases, hens were killed 24 hours after a single dose equal to the greater of either the LD50 or 5000 mg/kg. TOCP administration resulted in over 90% inhibition of brain neurotoxic esterase (NTE), but none of the other three compounds inhibited NTE to an extent (greater than 70%) which would be expected to result in OPIDN. Administration of TOCP, TBEP, or DBPP resulted in approximately a 70% decrease in plasma butyrylcholinesterase (BuChE) activity. TBP caused a 2-3 fold increase in BuChE activity. TBEP administration resulted in about 45% inhibition of acetycholinesterase (AChE) in brain. These results indicate that TBP, TBEP, and DBPP are all unlikely to cause OPIDN with any single sublethal dose.

Electrophysiologic changes following treatment with organophosphorus-induced delayed neuropathy-producing agents in the adult hen

Although clinical, pathological, and biochemical effects of organophosphorus-induced delayed neuropathy (OPIDN) have been intensively investigated in the adult hen, detailed electrophysiological studies are lacking. Adult white leghorn hens were treated with a single oral dose of either 30 mg/kg tri-2-cresyl phosphate (TOCP), 750 mg/kg TOCP, 4 mg/kg di- n-butyl-2,2-dichlorovinyl phosphate (DBCV), or 30 mg/kg di- n-butyl-2,2-dichlorovinyl phosphinate (DBCV-P). The 750 mg/kg TOCP and DBCV, but not the 30 mg/kg TOCP and DBCV-P, treatments resulted in clinical signs of OPIDN and mild to marked damage of the tibial nerve 21 days after dose. Twenty-four hr lymphocyte neurotoxic esterase (NTE) inhibition was used as an index of brain NTE inhibition for the various organophosphorus compound (OP) treatment. Twenty-four hr lymphocyte NTE inhibition for 30 mg/kg TOCP, 750 mg/kg TOCP, DBCV, and DBCV-P was 54.1, 87.1, 84.8, and 68.3%, respectively. Twenty-one days after dose, the TOCP-treated hens exhibited some abnormalities in conduction velocity and action potential duration in the tibial or sciatic nerves. No abnormalities were observed in action potential parameters of either the DBCV or DBCV-P treatments. Neurotoxic OP (TOCP and DBCV) treatment resulted in decreased refractoriness in the tibial nerve, increased refractoriness in the sciatic nerve, and elevated strength duration threshold for both nerves. These changes were not present in nerves from DBCV-P (a non-neurotoxic NTE inhibitor)-treated hens. These results suggest that refractory period and strength duration abnormalities in peripheral nerve correlated well with the production of OPIDN and are evident without coincident clinical signs or histopathology.

Lymphocyte and brain neurotoxic esterase: Dose and time dependence of inhibition in the hen examined with three organophosphorus esters

Certain organic phosphorus esters produce sensorimotor axonopathy in man and other species. There is an excellent correlation between the capacity of an organophosphorus compound to produce axonopathy and its ability to inhibit brain neurotoxic esterase (NTE) in hens. Because NTE is present in peripheral lymphocytes of both hen and man, it has been suggested that the lymphocyte enzyme might be useful both in experimental and clinical situations as an indicator of exposure to organophosphorus compounds producing axonopathy. Diethyl 4-nitrophenyl phosphate (paraoxon), tri-2-cresyl phosphate (TOCP), methyl 2,5-dichloro-4-bromophenyl phenylphosphonothionate (leptophos), and di- n-butyl-2,2-dichlorovinyl phosphate (di- n-butyl dichlorvos, DBDCV) were used to examine the relationship between lymphocyte and brain NTE inhibition in hens. As expected, paraoxon (0.75 mg/kg) did not inhibit NTE in brain or lymphocytes. TOCP (10 to 100 mg/kg), leptophos (25 to 150 mg/kg), and DBDCV (1.0 to 4.0 mg/kg) inhibited both brain and lymphocyte NTE activity in a doserelated manner with good correlation of inhibition between tissues taken 24 hr after exposure ( r 2 = 0.53 to 0.67; p < 0.020 to 0.001). However, correlation of inhibition between tissues taken from animals killed 48 hr after exposures was poor ( r 2 = 0.15 to 0.30; p < 0.10 to 0.05), with consistently less inhibition of lymphocyte NTE relative to brain NTE. This study indicates that assay of lymphocyte NTE can provide a good monitor of exposure to axonotoxic organophosphorus compounds within 24 hr between exposure and measurement.

Assessment of the delayed neurotoxic potential of isopropyl triphenylphosphate using a nontraditional testing strategy.

The potential of isopropyl triphenyl phosphate (ITP) to produce delayed neurotoxicity in hens was examined using several techniques. ITP contained O,O,O-triphenyl phosphate (24%), O-o-isopropylphenyl O,O-diphenyl phosphate (25%), O,O-diisopropyl-phenyl O-phenyl phosphate (20%), O-o, p-diisopropylphenyl O,O-diphenyl phosphate (18%) and O-p-isopropylphenyl O,O-diphenyl phosphate (6%). Hens treated twice, 3 wk apart, with doses of ITP as high as 11.7 g/kg showed no clinical signs of delayed neurotoxicity and only mild signs of general toxicity. Furthermore, none showed even subtle neurohistologic changes suggestive of delayed neurotoxicity. ITP produced dose-dependent inhibition of hen plasma cholinesterase and brain neurotoxic esterase (NTE). The study was continued because NTE inhibition has been shown to be a reliable predictor of organophosphates that produce delayed neurotoxicity. ITP was administered prior to tri-o-tolyl phosphate (TOCP) challenge in order to determine if it altered development of TOCP delayed neurotoxicity. ITP neither enhanced nor reduced the onset or severity of neurotoxicity produced by TOCP. The time-course for brain and spinal cord NTE inhibition by ITP and TOCP were compared and found to be different. The maximum brain NTE inhibition produced by ITP (doses up to 11.7 g/kg) was never complete (always less than 90%), and spinal cord NTE inhibition was significantly less than that produced in the brain. In contrast, brain and spinal cord inhibition produced by 500 mg TOCP/kg were equal and greater than 90%. This testing regimen showed that ITP produced an effect on NTE at the biochemical level without producing clinical or neurohistologic abnormalities in treated hens. Furthermore, this biochemical effect was qualitatively different than that produced by the delayed neurotoxicant TOCP.

Six-month daily treatment of sheep with neurotoxic organophosphorus compounds

The delayed neurotoxic effects of tri- o-cresyl-phosphate (TOCP), O-methyl- O-(4-bromo-2,5-dichlorophenyl) phenylphosphonothioate (leptophos), and O-ethyl O-(4-nitrophenyl) phenylphosphonothioate (EPN) at 5, 5, and 1 mg/kg/day, respectively, on male sheep were studied during 6 months of daily oral treatment under field conditions. A vehicle-control group of sheep given corn oil (0.1 ml/kg/day) only was used for comparison. All sheep were killed 24 h after the 180th daily treatment. Blood, brain, spinal cord, and sciatic nerve tissues were taken for histological and/or biochemical examinations. The results indicated that leptophos induced severe ataxia and paralysis in sheep following about 4 months of treatment. TOCP produced either mild ataxia or lameness in two of four sheep during the last week of experiment. On the other hand, none of the EPN-treated sheep showed clinical signs of neurotoxicity during the course of the experiment at the dosage tested. These clinical results were supported by histological findings and also by biochemical results with neurotoxic esterase (NTE) measurements. In the case of leptophos-treated sheep, numerous prominent degenerative lesions of axons were observed in spinal cords and brains. Similar but somewhat less numerous lesions were noted in sheep treated with TOCP. No histological changes were observed in similar tissues taken from EPN-treated sheep. The results also indicated that, for chronic exposure to these neurotoxic organophosphorus compounds in sheep, a threshold in excess of 60–70% prolonged inhibition of brain NTE, or 50–60% inhibition of spinal cord NTE must be exceeded to initiate clinical and/or histological neurotoxic effects.

Species susceptibility to delayed toxic neuropathy in relation to in vivo inhibition of neurotoxic esterase by neurotoxic organophosphorus esters.

Tri-o-cresyl phosphate (TOCP) and O-ethyl O-(4-cyanophenyl) phenylphosphonothioate (cyanofenphos, Surecide) were found to be delayed neurotoxicants. They were administered to chickens by gavage at 100 and 30 mg/kg . d for 15 d, respectively. In CD-1 mice neither TOCP nor cyanophenphos induced any of the usually recognized clinical symptoms of neuropathy when administered daily by gavage at 262 or 31.25 mg/kg . d for 30 d, respectively. In the chickens, TOCP and cyanofenphos produced about 98 and 90% in vivo inhibition of brain neurotoxic esterase (NTE) activity. In the mice, 24 h after the last daily dose, TOCP and cyanofenphos produced only about 50 and 40% in vivo inhibition of the brain NTE activity. Parathion [O,O-diethyl O-(4-nitrophenyl) phosphorothioate], at 2 or 6.75 mg/kg . d for 15 or 30 d, did not induce neuropathy in either chicken or mice and produced no significant in vivo inhibition of brain NTE activity at the end of the dosing regimen. The specific activity of NTE in control chicken brain crude homogenate was much higher than that in mouse brain homogenate. These results suggest that the differences between chickens and mice in susceptibility to neurotoxic organophosphates may be attributed to (1) inhibitor specificity of NTE forms in the brain in these two different animal species and/or (2) inability of the active metabolites of these neurotoxic compounds to reach the site of action.

Comparison of inhibitory activity of various organophosphorus compounds against acetylcholinesterase and neurotoxic esterase of hens with respect to delayed neurotoxicity

A variety of organophosphorus (OP) compounds with and without delayed neurotoxicity were examined for inhibitory power against neurotoxic esterase (NTE) and acetylcholinesterase (AchE) of hen brain in vitro and in vitro. Generally, delayed neurotoxicity induced by OP compounds correlated with high inhibition of NTE in vitro, whereas in vitro studies comparing I 50S for both enzymes did not provide a guide to evaluation of delayed neurotoxicity. Single oral administration of delayed neurotoxic EPN, leptophos and TOCP resulted in more than 80 per cent inhibition of brain NTE at neurotoxic doses, whereas non-delayed neurotoxic methyl parathion, fenitrothion and cyanophos caused weak inhibition at near lethal doses which gave rise to severe inhibition of brain AchE. A delayed neurotoxic dose of (−)-EPN caused more severe inhibition of brain NTE as compared with the same dose of the non-delayed neurotoxic ( + )-isomer. However, a few compounds produced severe inhibition of NTE at non-delayed neurotoxic doses. Hens paralysed by repeated administration of a low level of leptophos showed significant decreases in NTE activity of the brain and spinal cord.

The anomalous behaviour of dimethyl phosphates in the biochemical test for delayed neurotoxicity.

Several dimethyl phosphate behave anomalously in tests for delayed neurotoxicity. Doses given to hens caused high inhibition of brain neurotoxic esterase (NTE) but no ataxia. Less inhibition of NTE was seen in spinal cord than in brain. Di-isopropyl phosphorofluoridate caused equal inhibition of NTE in brain and cord. When dosing with dimethyl phosphates was repeated NTE inhibition in cord increased and pair-dosed birds became ataxic. In vitro brain and cord NTE were indistinguishable but the in vivo discrepancy between inhibition of brain and cord NTE was matched by a similar discrepancy in inhibition of AChE. It appears that ataxia arises from inhibition of spinal cord NTE and that only in the present cases (among about 200) was the effect in brain not a perfect biochemical monitor.