Triphenyl phosphite neurotoxicity in the hen: inhibition of neurotoxic esterase and of prophylaxis by phenylmethylsulfonyl fluoride

The neuropathic syndrome resulting in the cat and the rat from single or multiple doses of the phosphorous acid ester tiphenyl phosphite (TPP) has been reported to differ from the syndrome caused by numerous phosphoric acid esters, which is known as organophosphorous compound-induced delayed neurotoxicity (OPIDN). Since the hen is used to test compounds for OPIDN, we chose to study the neurotoxicity of single subcutaneous doses of TPP using this animal model. TPP (1000 mg/kg) produced progressive ataxia and paralysis which began to develop 5–10 days after dosing. Similar signs were observed when subcutaneous doses of the OPIDN-causing agents tri-o-cresyl phosphate (TOCP) or diisopropyl phosphorofluoridate (DFP) were administered. The minimum neurotoxic dose of TPP was 500 mg/kg. Prior administration of phenylmethylsulfonyl fluoride (PMSF) prevented the development of a neuropathy induced by DFP, but did not fully protect the hens from TPP or TOCP. PMSF slowed, but did not prevent, the neuropathy caused by TOCP. PMSF reduced the neurotoxicity of 500 mg/kg TPP, but increased the neurotoxicity of 1000 mg/kg TPP. TPP was found to be a very potent inhibitor of neurotoxic esterase (NTE), the putative target site for OPIDN, in vitro, with a k_i of about 2.1×10⁵ M^–1min^–1. Equimolar doses of either TPP (1000 mg/kg) and TOCP (1187 mg/kg) caused over 80% inhibition of neurotoxic esterase (NTE) in brain and sciatic nerve. This high level of NTE inhibition persisted for several weeks. This prolonged inhibition probably accounts for the inability of PMSF to block the neurotoxicity of TOCP. The dose-response curve for NTE inhibition 48 h after dosing indicated that a level of 70% inhibition correlated with the neurotoxicity of TPP.Subneurotoxic doses of TPP and DFP were found to have an additive effect which could be blocked by PMSF. These results indicate that TPP can cause OPIDN in the hen. The synergism between PMSF and the higher dose of TPP suggests the presence of a second neurotoxic effect as well.     

Potentiation of organophosphorus-induced delayed neurotoxicity by phenylmethylsulfonyl fluoride

It is well known that pretreatment with the serine esterase inhibitor phenylmethylsulfonyl fluoride (PMSF) can protect experimental animals from organophosphorus-induced delayed neurotoxicity (OPIDN), presumably by blocking the active site of neurotoxic esterase (NTE) such that binding and "aging" of the neuropathic OP is thwarted. We report here that while PMSF (60 mg/kg, sc) given 4 h before the neuropathic organophosphate (OP) mipafox (50 mg/kg, im) completely prevented the clinical expression of OPIDN in hens, the identical PMSF treatment markedly amplified the delayed neurotoxicity (relative to hens treated with OP only) if administered 4 h after mipafox (5 or 50 mg/kg, im). Moreover, in a separate experiment using diisopropylphosphorofluoridate (DFP) as the neurotoxicant in place of mipafox, posttreatment with PMSF 4 h after DFP (0.5 mg/kg) also accentuated the severity of ataxia. These data indicate that PMSF only protects against OPIDN if given prior to exposure to the neurotoxicant; treatment with PMSF after OP exposure critically exacerbates the delayed neurotoxicity from exposure to organophosphorus compounds.     

Neurotoxicity induced in differentiated SK-N-SH-SY5Y human neuroblastoma cells by organophosphorus compounds

Organophosphorus (OP) compounds used as insecticides and chemical warfare agents are known to cause potent neurotoxic effects in humans and animals. Organophosphorus-induced delayed neuropathy (OPIDN) is currently thought to result from inhibition of neurotoxic esterase (NTE), but the actual molecular and cellular events leading to the development of OPIDN have not been characterized. This investigation examined the effects of OP compounds on the SY5Y human neuroblastoma cells at the cellular level to further characterize cellular targets of OP neurotoxicity. Mipafox and paraoxon were used as OP models that respectively do and do not induce OPIDN. Mipafox (0.05 mM) significantly decreased neurite length in SY5Y cells differentiated with nerve growth factor (NGF) while paraoxon at the same concentration had no effect when evaluated after each of three 4-day developmental windows during which cells were treated daily with OP or vehicle. In contrast, paraoxon but not mipafox altered intracellular calcium ion levels ([Ca(2+)](i)), as seen in three types of experiments. First, immediately following the addition of a single high concentration of OP to the culture, paraoxon caused a transient increase in [Ca(2+)](i), while mipafox up to 2 mM had no effect. Paraoxon hydrolysis products could also increase intracellular Ca(2+) levels, although the pattern of rise was different than it appeared immediately after paraoxon administration. Second, repeated low-level paraoxon treatment (0.05 mM/day for 4 days) decreased basal [Ca(2+)](i) in NGF-differentiated cells, though mipafox had no effect. Third, carbachol, a muscarinic acetylcholine receptor agonist, transiently increased [Ca(2+)](i) in differentiated cells, an affect attenuated by 4-day pretreatment with paraoxon (0.05 mM/day), but not by pretreatment with mipafox. These results indicate that the decrease in neurite extension that resulted from mipafox treatment was not caused by a disruption of Ca(2+) homeostasis. The effects of OPs that cause or do not cause OPIDN were clearly distinguishable, not only by their effects on neurite length, but also by their effects on Ca(2+) homeostasis in differentiated SY5Y cells.     

The homeostasis of phosphatidylcholine and lysophosphatidylcholine was not disrupted during tri-o-cresyl phosphate-induced delayed neurotoxicity in hens

Little is known regarding early biochemical events in organophosphate-induced delayed neurotoxicity (OPIDN) except for the essential inhibition of neuropathy target esterase (NTE). We hypothesized that the homeostasis of lysophosphatidylcholine (LPC) and/or phosphatidylcholine (PC) in nervous tissues might be disrupted after exposure to the organophosphates (OP) which participates in the progression of OPIDN because new clues to possible mechanisms of OPIDN have recently been discovered that NTE acts as lysophospholipase (LysoPLA) in mice and phospholipase B (PLB) in cultured mammalian cells. To bioassay for such phospholipids, we induced OPIDN in hens using tri-o-cresyl phosphate (TOCP) as an inducer with phenylmethylsulfonyl fluoride (PMSF) as a negative control; and the effects on the activities of NTE, LysoPLA and PLB, the levels of PC, LPC, and glycerophosphocholine (GPC), and the aging of NTE enzyme in the brain, spinal cord, and sciatic nerves were examined. The results demonstrated that the activities of NTE, NTE-LysoPLA, LysoPLA, NTE-PLB and PLB were significantly inhibited in both TOCP- and PMSF-treated hens. The inhibition of NTE and NTE-LysoPLA or NTE-PLB showed a high correlation coefficient in the nervous tissues. Moreover, the NTE inhibited by TOCP was of the aged type, while nearly all of the NTE inhibited by PMSF was of the unaged type. No significant change in PC or LPC levels was observed, while the GPC level was significantly decreased. However, there is no relationship found between the GPC level and the delayed symptoms or aging of NTE. All results suggested that LPC and/or PC homeostasis disruption may not be a mechanism for OPIDN because the PC and LPC homeostasis was not disrupted after exposure to the neuropathic OP, although NTE, LysoPLA, and PLB were significantly inhibited and the GPC level was remarkably decreased.     

Clinical expression of organophosphate-induced delayed polyneuropathy in rats.

Single doses of certain organophosphates (OP), such as dibutyl-2,2-dichlorovinyl phosphate (DBDCVP) cause organophosphate-induced delayed polyneuropathy (OPIDP) in hens. Clinical effects correlate with inhibition of neuropathy target esterase (NTE) which is considered the target for this toxicity. Pre-treatment with non-neuropathic NTE inhibitors, such as phenylmethanesulfonyl fluoride (PMSF), protects from OPIDP. However, when given after OPs, these compounds promote OPIDP. Chicks are relatively resistant to OPIDP despite high NTE inhibition. It has also always been reported that rats represent a species which is resistant to OPIDP and that they might develop morphological but not clinical signs of OPIDP. We report here that clinical OPIDP can be produced in 3.5- and 6-month-old rats by DBDCVP (5 mg/kg s.c.) and that it correlates with high (> 90%) NTE inhibition. When PMSF (120 mg/kg s.c. x 2) was given after DBDCVP, OPIDP was promoted. Pretreatment with PMSF protected from OPIDP. We conclude that resistance to OPIDP in the rat is age-related, as it is in the hen.     

The relationship between neurological damage and neurotoxic esterase inhibition in rats acutely exposed to tri-ortho-cresyl phosphate

A rodent model of organophosphorus-induced delayed neuropathy (OPIDN) has been developed using Long-Evans adult male rats exposed to tri-ortho-cresyl phosphate (TOCP). In the present study an attempt was made to relate neurochemical with neuropathological changes in rats exposed to single dosages of TOCP ranging from 145 to 3480 mg/kg. The degree of neurotoxic esterase (NTE) inhibition, measured at 20 and 44 hr and at 14 days postexposure was correlated with the appearance of spinal cord pathology 14 days postexposure in a separate group of similarly dosed rats. Those dosages that inhibited mean NTE activity in spinal cord greater than or equal to 72% and brain greater than or equal to 66% of control values within 44 hr postexposure produced marked spinal cord pathology 14 days postexposure in greater than or equal to 90% of similarly dosed animals. In contrast, dosages of TOCP which inhibited mean NTE activity in the spinal cord less than or equal to 65% and in the brain less than or equal to 57% produced spinal cord pathology in less than or equal to 15% of the animals. These data indicate that NTE inhibition may be used as a biochemical predictor for TOCP-induced neurological damage in rats.     

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 correlate well with the production of OPIDN and are evident without coincident clinical signs or histopathology.     

Triphenyl phosphite: in vivo and in vitro inhibition of rat neurotoxic esterase

Organophosphorus compounds which, after acute administration, inhibit neurotoxic esterase (NTE) by greater than or equal to 65% and undergo a subsequent "aging" reaction, produce a delayed neuropathy characterized by degeneration of large and long nerve fibers (OPIDN). The present studies examine in detail the NTE-inhibiting properties of triphenyl phosphite (TPP), a plasticizer which produces ataxia and degeneration of the spinal cord in animals. A neurotoxic dosing regimen (1184 mg/kg/week, sc, for 2 weeks) inhibited both brain and spinal cord NTE (less than or equal to 40%) only marginally 4 and 48 hr postdosing. By contrast, TPP was shown in vitro to be a potent (150 = 0.98 microM) inhibitor of rat brain NTE relative to Mipafox or diisopropyl phosphorofluoridate. Compounds structurally related to TPP (i.e., triphenyl phosphate, triphenyl phosphine, trimethyl phosphite, and phenol) failed to inhibit NTE in vitro at less than 10 microM concentrations. Close examination of the TPP inhibition of NTE showed a nonlinear relationship between the duration of incubation time and loss of log(NTE activity). Preincubation of 10 microM TPP in buffer (37 degrees C) resulted in a time-dependent loss of TPP's ability to inhibit NTE. In summary, TPP is a powerful NTE inhibitor in vitro, but only a marginal NTE inhibitor after in vivo administration. These results raise questions as to the causal events mediating TPP-induced neuropathy in the rat.     

Relative inhibitory potencies of chlorpyrifos oxon,chlorpyrifos methyl oxon,and mipafox for acetylcholinesterase versus neuropathy target esterase

The relative inhibitory potency (RIP) of an organophosphorus (OP) inhibitor against acetylcholinesterase (AChE) versus neuropathy target esterase (NTE) may be defined as the ratio [k(i)(AChE)/k(i)(NTE)], where k(i) is the bimolecular rate constant of inhibition for a given inhibitor against each enzyme. RIPs greater than 1 correlate with the inability of ageable OP inhibitors or their parent compounds to produce OP compound-induced delayed neurotoxicity (OPIDN) at doses below the LD50. The RIP for chlorpyrifos oxon (CPO) is >1 for enzymes from hen brain homogenate, and the parent compound, chlorpyrifos (CPS), cannot produce OPIDN in hens at sublethal doses. This study was carried out to test the hypothesis that the RIP for the methyl homologue of CPO, chlorpyrifos methyl oxon (CPMO), is >1 and greater than the RIP for CPO. Mipafox (MIP), an OP compound known to produce OPIDN, was included for comparison. Hen brain microsomes were used as the enzyme source, and k(i) values (mean +/- SE, microM(-1) min(-1)) were determined for AChE and NTE (n = 3 and 4 separate experiments, respectively). The k(i) values for CPO, CPMO, and MIP against AChE were 17.8 +/- 0.3, 10.9 +/- 0.1, and 0.00429 +/- 0.00001, respectively, and for NTE were 0.0993 +/- 0.0049, 0.0582 +/- 0.0013, and 0.00498 +/- 0.00006, respectively. Corresponding RIPs for CPO, CPMO, and MIP were 179 +/- 9, 187 +/- 4, and 0.861 +/- 0.011, respectively. The results demonstrate that RIPs for CPO and CPMO are comparable, markedly different from that for MIP, and >1, indicating that CPS methyl, like CPS, could not cause OPIDN at sublethal doses.     

Comparative studies of two organophosphorus compounds in the mouse

A rodent model, the albino mouse, was used to investigate the in vitro and in vivo capacity of 2 organophosphate (OP) compounds, mipafox and ecothiopate, to inhibit enzymes considered to be involved in the mechanisms of OP toxicity. Mipafox and ecothiopate were chosen as model compounds because the former can produce a delayed neuropathy whereas the latter does not. Mipafox (110 μmol/kg s.c.) inhibited brain acetylcholinesterase (AChE), neuropathy target esterase (NTE) and phenylvalerate hydrolases by 58, 64 and 65%, while diaphragm AChE and phenylvalerate hydrolases were inhibited by 66 and 80%, respectively. In contrast, ecothiopate (0.5 μmol/kg) had no effect on brain NTE or on brain or diaphragm phenylvalerate hydrolases. At the same time, diaphragm AChE was inhibited by 60% while brain AChE activity had increased by 15% of control. Mipafox was a potent inhibitor of AChE and NTE in vitro. Although ecothiopate was a highly potent anti-ChE in vitro, it had no inhibitory effect on NTE.     

Neurofilament 200 as an indicator of differences between mipafox and paraoxon sensitivity in Sy5Y neuroblastoma cells

Organophosphorus (OP) compounds produce potent neurotoxic effects in humans, including organophosphorus-induced delayed neuropathy (OPIDN). This investigation examined the potential for the 200-kD neurofilament protein (NF200) and other neuronal proteins to serve as indicators for neurite damage in a differentiated SY5Y human neuroblastoma cell culture system. Mipafox, which induces OPIDN, increased NF200 protein expression in SY5Y cells differentiated with human recombinant beta-nerve growth factor (NGF, 20 ng/ml) in a concentration-dependent manner, compared to NGF controls, when SY5Y cells were exposed to 0.3 or 30 microM mipafox during the last 5 days of neurite extension (experimental set A). However, mipafox produced little change in NF200 protein expression in SY5Y cells exposed continuously throughout neurite elongation (experimental set B). Paraoxon (up to 30 microM), which does not produce OPIDN, did not produce any change in NF200 expression in set A or set B. The upregulation of NF200 by mipafox may represent a compensatory response to neurite degeneration. Two other neuronal proteins, growth-associated protein 43 (GAP43) and microtubule-associated protein 2ab (MAP2ab), showed no changes in response to OP treatment in NGF-treated cells. Protein expression of NF200 was shown to be an indicator by which the sensitivities of SY5Y cells to mipafox and paraoxon were distinguishable at the molecular level. These results indicate an alternative approach and test system for investigating structure-activity relationships of OPs.     

Further studies toward a mouse model for biochemical assessment of neuropathic potential of organophosphorus compounds

Inhibition and aging of neuropathy target esterase (NTE) by neuropathic organophosphorus (OP) compounds triggers OP compound‐induced delayed neuropathy (OPIDN), whereas inhibition of acetylcholinesterase (AChE) produces cholinergic toxicity. The neuropathic potential of an OP compound is defined by its relative inhibitory potency toward NTE vs. AChE assessed by enzyme assays following dosing in vivo or after incubations of direct‐acting compounds or active metabolites with enzymes in vitro. The standard animal model of OPIDN is the adult hen, but its large size and high husbandry costs make this species a burdensome model for assessing neuropathic potential. Although the mouse does not readily exhibit clinical signs of OPIDN, it displays axonal lesions and expresses brain AChE and NTE. Therefore, the present research was performed as a further test of the hypothesis that inhibition of mouse brain AChE and NTE could be used to assess neuropathic potential using mouse brain preparations in vitro or employing mouse brain assays following dosing of OP compounds in vivo. Excellent correlations were obtained for inhibition kinetics in vitro of mouse brain enzymes vs. hen brain and human recombinant enzymes. Furthermore, inhibition of mouse brain AChE and NTE after dosing with OP compounds afforded ED₅₀ ratios that agreed with relative inhibitory potencies assessed in vitro. Taken together, results with mouse brain enzymes demonstrated consistent correspondence between in vitro and in vivo predictors of neuropathic potential, thus adding to previous studies supporting the validity of a mouse model for biochemical assessment of the ability of OP compounds to produce OPIDN. Copyright © 2014 John Wiley & Sons, Ltd.     

Potentiation of Organophosphorus-Induced Delayed Neurotoxicity Following Phenyl Saligenin Phosphate Exposures in 2-, 5-, and 8-Week-Old Chickens

Phenylmethylsulfonyl fluoride (PMSF), a nonneuropathic inhibitorof neurotoxk esterase (NTE), is a known potentiator of organophosphorus-induceddelayed neurotoxicity (OPIDN)- The ability of PMSF posttreatment(90 mg/kg, sc, 4 hr after the last PSP injection) to modifydevelopment of delayed neurotoxicity was examined in 2-, 5-,and 8-week-old White Leghorn chickens treated either one, two,or three times (doses separated by 24 hr) with the neuropathicOP compound phenyl saligenin phosphate (PSP, 5 mg/kg, sc). NTEactivity was measured in the cervical spinal cord 4 hr afterthe last PSP treatment. Development of delayed neurotoxicitywas measured over a 16-day postexposure period. All PSP-treatedgroups exhibited >97% NTE inhibition regardless of age ornumber of OP treatments. Two-week-old birds did not developclinical signs of neurotoxicity in response to either singleor repeated OP treatment regimens nor following subsequent treatmentwith PMSF. Five-week-old birds were resistant to the clinicaleffects of a single PSP exposure and were minimally affectedby repeated doses. PMSF posttreatment, however, significantlyamplified the clinical effects of one, two, or three doses ofPSP. A single exposure to PSP induced slight to moderate signsof delayed neurotoxicity in 8-week-old birds with more extensiveneurotoxicity being noted following repeated dosing. As with5-week-old birds, PMSF exacerbated the clinical signs of neurotoxicitywhen given after one, two, or three doses of PSP in 8-week-oldbirds. Axonal degeneration studies supported the clinical findings:PMSF posttreatment did not influence the degree of degenerationin 2-week-old chickens but resulted in more severe degeneration(relative to PSP only exposure) in cervical cords from both5- and 8-week-old birds. The results indicate that PMSF doesnot alter the progression of delayed neurotoxicity in very young(2 weeks of age) chickens but potentiates PSP-induced delayedneurotoxicity in the presence of 0–3% residual NTE activityin older animals. We conclude that posttreatment with neuropathicor nonneuropathic NTE inhibitors, following virtually completeNTE inhibition by either single or repeated doses of a neuropathicagent in sensitive age groups, can modify both the clinicaland morphological indices of delayed neurotoxicity. This studyfurther supports the hypothesis that potentiation of OPIDN occursthrough a mechanism unrelated to NTE.     

Kinetic interactions of a neuropathy potentiator (phenylmethylsulfonyl fluoride) with the neuropathy target esterase and other membrane bound esterases

Phenylmethylsulfonyl fluoride (PMSF) is a protease and esterase inhibitor that causes protection, or potentiation/“promotion,” of organophosphorus delayed neuropathy (OPIDN), depending on whether it is dosed before or after an inducer of delayed neuropathy, such as mipafox. The molecular target of the potentiation/promotion of OPIDN has not yet been identified. The kinetic data of phenyl valerate esterase inhibition by PMSF were obtained with membrane chicken brain fractions, the animal model and tissue in which neuropathy target esterase (NTE) was first described. Data were analyzed using a kinetic model with a multienzymatic system in which inhibition, simultaneous chemical hydrolysis of the inhibitor and “ongoing inhibition” (inhibition during the substrate reaction) were considered. Three main esterase components were discriminated: two sensitive enzymatic entities representing 44 and 41 %, with I ₅₀ (20 min) of 35 and 198 μM at 37 °C, respectively, and a resistant fraction of 15 % of activity. The estimated constant of the chemical hydrolysis of PMSF was also calculated (kh = 0.28 min^?1). Four esterase components were globally identified considering also previously data with paraoxon and mipafox: EPα (4–8 %), highly sensitive to paraoxon and mipafox, spontaneously reactivates after inhibition with paraoxon, and resistant to PMSF; EPβ (38–41 %), sensitive to paraoxon and PMSF, but practically resistant to mipafox, this esterase component has the kinetic characteristics expected for the PMSF potentiator target, even though paraoxon cannot be a potentiator in vivo due to high AChE inhibition; EPγ (NTE) (39–48 %), paraoxon-resistant and sensitive to the micromolar concentration of mipafox and PMSF; and EPδ (10 %), resistant to all the inhibitors assayed. This kinetic characterization study is needed for further isolation and molecular characterization studies, and these PMSF phenyl valerate esterase components will have to be considered in further studies of OPIDN promotion. A simple test for monitoring the four esterase components is proposed.     

Triphenylphosphite neuropathy in hens

Single doses of triphenyl phosphite (TPP), a triester of trivalent phosphorus, cause ataxia and paralysis in hens. Characteristics of neurotoxicity were described as somewhat different from organophosphate induced delayed polyneuropathy (OPIDP), which is caused by triesters of pentavalent phosphorus. The onset of TPP neuropathy was reported to occur earlier than that of OPIDP (5–10 versus 7–14 days after dosing, respectively), and chromatolysis, neuronal necrosis and lesions in certain areas of the brain were found in TPP neuropathy only. Pretreatment with phenylmethanesulfonyl fluoride (PMSF) protects from OPIDP, but it either partially protected from effects of low doses or exacerbated those of higher doses of TPP. In order to account for these differences with OPIDP, it was suggested that TPP neuropathy results from the combination of two independent mechanisms of toxicity: typical OPIDP due to inhibition of neuropathy target esterase (NTE) plus a second neurotoxicity related with other target(s). We explored TPP neuropathy in the hen with attention to the phenomena of promotion and protection which are both caused by PMSF when given in combination with typical neuropathic OPs. When PMSF is given before neuropathic OPs it protects from OPIDP; when given afterwards it exaggerates OPIDP. The former effect is due to interactions with NTE, the latter to interactions with an unknown site. The time course of NTE reappearance after TPP (60 or 90 mg/kg i.v.) inhibition showed a longer half-life when compared to that after PMSF (30 mg/kg s.c.) (10–15 versus 4–6 days, respectively). The clinical signs of TPP neuropathy (60 or 90 mg/kg i.v.) were similar to those observed in OPIDP, appeared 7–12 days after treatment, correlated with more than 70% NTE inhibition/aging and were preceded by a reduction of retrograde axonal transport in sciatic nerve of hens. TPP (60 mg/kg i.v.) neuropathy was promoted by PMSF (120 mg/kg s.c.) given up to 12 days afterwards and was partially protected by PMSF (10–120 mg/kg s.c.) when given 24 h before TPP (60 or 90 mg/kg i.v.). The previously reported early onset of TPP neuropathy might be related to the higher dose used in those experiments and to the resulting more severe neuropathy. The lack of full protection might be explained by the slow kinetics of TPP, which would cause substantial NTE inhibition when PMSF effects on NTE had subsided. Since PMSF also affects the promotion site when given before initiation of neuropathy, the resulting neuropathy would then be due to both protection from and promotion of TPP effects by PMSF. No promotion by PMSF (120 mg/kg s.c.) was observed in TPP neuropathy (90 mg/kg i.v.) partially protected by PMSF (10–30 mg/kg s.c.) This might also be explained by the concurrent effects on NTE and on the promotion site obtained with PMSF pretreatment. We conclude that TPP neuropathy in the hen is likely to be the same as typical OPIDP. The unusual effects of combined treatment to hens with TPP and PMSF are explained by the prolonged pharmacokinetics of TPP and by the dual effect of PMSF i.e. protection from and promotion of OPIDP.     

Effects of fenthion, fenitrothion and desbromoleptophos on gait, acetylcholinesterase, and neurotoxic esterase in young chicks after in ovo exposure

Previous studies have demonstrated that gait is affected in chicks exposed to organophosphorus esters (OPs) that induce delayed neurotoxicity (OPIDN) in adult hens. To investigate the developmental relationship between such functional deficits and OPIDN, chicks were exposed to 3 OPs with different OPIDN potential. Desbromoleptophos (DBL) induces OPIDN in adult hens; fenthion (FEN) has uncertain OPIDN potential; fenitrothion (FTR) does not induce OPIDN. Chicks were treated by injection into the egg on day 15 of incubation, after the presumed period of OP-induced structural teratogenesis. AChE and neurotoxic esterase (NTE) were assayed during incubation and in parallel with post-hatching evaluations of gait. DBL, 125 mg/kg in ovo, caused paralysis in 70% of chicks after hatching. The gait of surviving chicks was affected for at least 6 weeks and marked by toes curling under. NTE was inhibited until 10 days post-hatching and AChE until hatching. FEN did not inhibit NTE significantly, but AChE was significantly inhibited until hatching. Chicks exposed as embryos to FEN were hyperactive and aggressive. Gait was still affected 6 weeks after treatment with 3 mg/kg FEN. FTR at 125 mg/kg inhibited AChE until day 10 post-hatching, but neither inhibited NTE nor affected gait. The growth of OP-exposed chicks was not significantly decreased, so the decreased length and increased width of the stride could not be ascribed to stunted growth. We conclude that OPs cause irreversible effects on gait that are not related to their defined neurotoxic effects, since altered gait (1) occurs below the age of sensitivity to OPIDN, (2) is seen in the absence of NTE inhibition and (3) does not invariably accompany AChE inhibition.     

Isofenphos and an in vitro activation assay for delayed neuropathic potential

Organophosphorus compounds (OPs) that cause organophosphorus ester-induced delayed neuropathy (OPIDN) generally inhibit neurotoxic esterase (NTE). However, the assay itself, when conducted in vitro, misses OPs that are activated into OPIDN-causing agents in the body. A preparation of liver mixed-function oxidases and brain NTE was used to rapidly detect activations of OPs. The compounds (0.1 mM or less) to be tested were incubated with microsomes isolated from livers of phenobarbital-treated chick embryos (P-450 content averaged 1.81 +/- 0.27 nmol/mg protein, means +/- SD, N = 5) and NTE (average of 13.8 nmol/min/mg protein) from untreated chick embryo brains. The NTE was separated by calcium precipitation and its activity assayed as usual. The low inhibitions of NTE of compounds that were not neurotoxic (parathion, Diazinon) did not increase in the presence of NADPH; inhibitions of NTE of compounds that required activation (leptophos, S,S,S-tri-n-butyl phosphorotrithioate, and tri-o-cresyl phosphate) greatly increased with NADPH. Both the recently identified neuropathic OP isofenphos (IFP) and its oxon required activation to inhibit NTE (inhibitions of 20 and 80%, respectively). Evidence is presented that the possible neuropathic metabolite is des-N-isopropyl IFP oxon.     

Neuropathy target esterase in mouse whole blood as a biomarker of exposure to neuropathic organophosphorus compounds

The adult hen is the standard animal model for testing organophosphorus (OP) compounds for organophosphorus compound‐induced delayed neurotoxicity (OPIDN). Recently, we developed a mouse model for biochemical assessment of the neuropathic potential of OP compounds based on brain neuropathy target esterase (NTE) and acetylcholinesterase (AChE) inhibition. We carried out the present work to further develop the mouse model by testing the hypothesis that whole blood NTE inhibition could be used as a biochemical marker for exposure to neuropathic OP compounds. Because brain NTE and AChE inhibition are biomarkers of OPIDN and acute cholinergic toxicity, respectively, we compared NTE and AChE 20‐min IC₅₀ values as well as ED₅₀ values 1 h after single intraperitoneal (i.p.) injections of increasing doses of two neuropathic OP compounds that differed in acute toxicity potency. We found good agreement between the brain and blood for in vitro sensitivity of each enzyme as well for the ratios IC₅₀(AChE)/IC₅₀(NTE). Both OP compounds inhibited AChE and NTE in the mouse brain and blood dose‐dependently, and brain and blood inhibitions in vivo were well correlated for each enzyme. For both OP compounds, the ratio ED₅₀(AChE)/ED₅₀(NTE) in blood corresponded to that in the brain despite the somewhat higher sensitivity of blood enzymes. Thus, our results indicate that mouse blood NTE could serve as a biomarker of exposure to neuropathic OP compounds. Moreover, the data suggest that relative inhibition of blood NTE and AChE provide a way to assess the likelihood that OP compound exposure in a susceptible species would produce cholinergic and/or delayed neuropathic effects. Copyright © 2016 John Wiley & Sons, Ltd.     

Repeated low doses of O-(2-chloro-2,3,3 trifluorocyclobutyl) O-ethyl S-propyl phosphorothioate (KBR-2822) do not cause neuropathy in hens

Certain esterase inhibitors such as O-(2-chlo-ro-2,3,3-trifluorocyclobutyl) O-ethyl S-propyl phosphorothioate (KBR-2822) and phenylmethanesulfonyl fluoride (PMSF) cause exacerbation (promotion) of toxic and traumatic axonopathies. Although these chemicals are capable of inhibiting neuropathy target esterase (NTE), which is the target for organophosphate induced delayed neuropathy, the target for promotion is unlikely to be NTE. Experiments were aimed to ascertain if neuropathy is caused by repeated dosing with a promoter not causing NTE inhibition and in the absence of deliberate injury to axons. Hens were treated with KBR-2822 (0.2 or 0.4 mg/kg per day) by gavage for 90 days and observed for clinical signs up to 21–23 days after treatment when histopathological examination was carried out. NTE and acetylcholinesterase (AChE) were measured at intervals and mean percentages of inhibition at steady state of inhibition/resynthesis (on day 20) were as follows: mean inhibition NTE was ≤8% in the 0.2 mg/kg group and between 15 and 18% in the 0.4 mg/kg group in brain, spinal cord and peripheral nerve; mean AChE inhibition in brain was 31 and 57% in the two experimental groups, respectively. Controls treated with paraoxon (not neuropathic or a promoter and given at 0.05 mg/kg per day by gavage) showed 45% mean AChE inhibition and no NTE inhibition. Neither clinical nor morphological signs of neuropathy were observed in any group. To ascertain whether sub-clinical lesions were produced by the repeated treatment with KBR-2822, hens were given KBR-2822 (0.2 mg/kg per day) for 21 days by gavage followed by PMSF (120 mg/kg s.c. 24 h after the last dose of KBR-2822). A control group of hens was treated with the neuropathic DFP (0.03 mg/kg s.c. daily for 21 days causing 40–50% NTE inhibition) followed by PMSF (120 mg/kg s.c.). After PMSF, the KBR-2822 treated hens did not develop neuropathy whereas DFP treated hens did. Lack of neuropathy after repeated treatment with KBR-2822 indicates that a continuous promoting `pressure' on hen axons is harmless in the absence of a concurrent biochemical or neurotoxic injury. Received: 22 May 1997 / Accepted: 16 September 1997     

Organophosphate induced delayed neuropathy in genetically dissimilar chickens: studies with tri-ortho-cresyl phosphate (TOCP) and trichlorfon

Measurements of plasma cholinesterase (pl.ChE), brain cholinesterase (Br.ChE) and brain Neuropathy Target Esterase (Br.NTE) were made in three different lineages of chickens. All birds received toxicants through gavage in a single oral dose between 08:00 and 09:00 h, after overnight fast. Babcock chickens were treated with 800 mg/kg tri-ortho-cresyl phosphate (TOCP) or 80 mg/kg trichlorfon. The TOCP group had 82% Br.NTE inhibition, when compared to the control group, and no birds displayed symptoms of clinical organophosphate-induced delayed neuropathy (OPIDN). Hy-line w36 lineage chickens were given 1600 mg/kg TOCP and despite this higher dose, Br.NTE inhibition was similar that presented by Babcock chickens. Isabrown chickens were given 1600 mg/kg TOCP or 80 mg/kg trichlorfon. At 36 h all trichlorfon treated birds had from 80 to 90% inhibition of Pl.ChE and Br.ChE, when compared to controls. However, Br.NTE was inhibited less than 20%, and there were no clinical signs of OPIDN. All TOCP treated isabrown chickens had more than 80% Br.NTE inhibition while one of them exhibited just light signs of OPIDN, two chickens became totally paralyzed. This finding suggested that chicken strain was important in the appearance of OPIDN. In addition, 70-80% of NTE inhibition was necessary but was not sufficient to produce OPIDN in chickens, since babcock and hy-line w36 chickens exhibited NTE inhibition in the range of 70-80% without clinical signs of OPIDN.