Inhibitor Report for: Tri-o-cresylphosphate

The restoration of plasma acetylcholinesterase activity in mice following inhibition by organophosphorus pesticides and nerve agents has been attributed to synthesis of new enzyme. It is generally assumed that activity levels return to normal, are stable and do not exceed the normal level. We have observed over the past 10 years that recovery of acetylcholinesterase activity levels in mice treated with organophosphorus agents (OP) exceeds pretreatment levels and remains elevated for up to 2 months. The most dramatic case was in mice treated with tri-cresyl phosphate and tri-ortho-cresyl phosphate, where plasma acetylcholinesterase activity rebounded to a level 250% higher than the pretreatment activity. The present report summarizes our observations on plasma acetylcholinesterase activity in mice treated with chlorpyrifos, chlorpyrifos oxon, diazinon, tri-ortho-cresyl phosphate, tri-cresyl phosphate, tabun thiocholine, parathion, dichlorvos, and diisopropylfluorophosphate. We have developed a hypothesis to explain the excess acetylcholinesterase activity, based on published observations. We hypothesize that acetylcholinesterase activity is induced when cells undergo apoptosis and that consequently there is a rise in the level of plasma acetylcholinesterase.

Nine esterase fractions hydrolyzing 1-naphthylacetate were revealed in Triton X-100-solubilized extracts from aphides homogenates by polyacrylamide gel electrophoresis. The less mobile fractions 1-4 were identified as cholinesterases, using specific inhibitors--eserine and the cationic phosphoorganic inhibitor Gd-42; fractions 5-7 were related to carboxylesterases, using specific inhibition by triorthocresylphosphate and O,O-dimethyl (2,2-dichlorvinyl)phosphate. The most mobile fractions 8-9 which were resistant to the inhibitors, were classified as arylesterases. The aphis cholinesterase fractions revealed the highest mobility; the activity of carboxylesterase fractions was lower. Thiophosphonate--C8H17O(CH3)P(O)-SCH2SCH2COOCH3 was found to be a highly efficient selective inhibitor of aphis carboxylesterase, i. e. the kII values for carboxylesterase and cholinesterase were equal to 10(8) and 10(5) M-1 min-1, respectively. The thiophosphoorganic derivatives containing a beta-alanine residue in the cleaved part are more specific to acetylcholinesterase and carboxylesterase than those containing a valine residue. Studies with enanthiomers--C2H5O(CH3)P(O)SCH2CONHCH2CH2COOC2H5 and (C2H5O)2P(O)SCH2CONHCH(iC3H7)COOC2H5 have demonstrated that the asymmetry due to the central phosphorus atom is more essential for the acetylcholinesterase and carboxylesterase activities than that connected with the carbon atom in the cleaved part of the inhibitor molecule. During the interaction of the enanthiomers with the asymmetric phosphorus the stereospecificity of acetylcholinesterase is much higher than that of carboxylesterase. In terms of stereospecificity of the esterase site aphis acetylcholinesterase is is similar to its mammalian counterpart, while carboxylesterase from the same source is rather close to mammalian butyrylcholinesterase.

Ten day old chick sympathetic ganglia cultured in a microslide assembly were treated with a selected group of organophosphate pesticides to evaluate their cytotoxicity ranges, and the usefulness of such a model for screening pesticides. Examination by phase contrast and light microscopy for chemically-induced morphological alteration of nerve fibers, glial cells and neurons provided the criteria for quantitation and assessment of the toxic effects. Concentrations that produced half-maximal effects ranged from 1 x 10(-6)M (severely toxic) for methylparathian, diazinon, paraoxon, mevinphos, diisopropylfluorophosphate, tri-o-tolyl phosphate and its mixed isomers to a 1 x 10(-3)M (intermediate) for malathion, leptophos, coumaphos, mono- and dicrotophos. Some or no effects were evident at 1 x 10(2-)M for O'ethyl-O-p-nitrophenyl phenyl phosphonothioate, tri-m-tolylphosphate, chlorpyriphos and triphenyl phosphate. In all instances, nerve fibers were more sensitive than neurons or glial cells to insecticides. All cellular growth was inhibited at 1 x 10(-2)M (except triphenyl phosphate). Below 1 x 10(-7)M, no inhibitory effects were evident. The secondary abnormalities included decreased cellular migration, diffuse cellular growth pattern, increased vacuolization, nerve fiber swelling and cellular degeneration. The cytotoxic effects of these chemicals do not appear to be related to in vivo toxicity or cholinesterase inhibition potential.

Tricresyl phosphates (TCPs), as representative aromatic organophosphate flame retardants (OPFRs), have received much attention due to their potential neurotoxicity and endocrine-disrupting effects. However, the role of estrogen receptor alpha (ERalpha) and G protein-coupled estrogen receptor (GPER) in their estrogen disrupting effects remains poorly understood. Therefore, in this study, three TCP isomers, tri-o-cresyl phosphate (ToCP), tri-m-cresyl phosphate (TmCP) and tri-p-cresyl phosphate (TpCP), were examined for their activities on ERalpha by using two-hybrid yeast assay, and action on GPER by using Boyden chamber assay, cAMP production assay, calcium mobilization assay and molecular docking analysis. The results showed that three TCP isomers were found to act as ERalpha antagonists. Conversely, they had agonistic activity on GPER to promote GPER-mediated cell migration of MCF7 cells and SKBR3 cells. Both ToCP and TpCP activated GPER-mediated cAMP production and calcium mobilization, whereas TmCP had different mode of action, it only triggered GPER-mediated calcium mobilization, as evidenced by using the specific GPER inhibitor (G15) and GPER overexpressing experiments. Molecular docking further revealed that the way of interaction of TmCP and TpCP with GPER was different from that of ToCP with GPER, and higher activity of ToCP in activating GPER-mediated pathways might be associated with the alkyl substitution at the ortho position of the aromatic ring. Our results, for the first time, found a new target, GPER, for TCPs exerting their estrogen-disrupting effects, and demonstrated complex estrogen-disrupting effects of three TCP isomers involved their opposite activities toward ERalpha and GPER.

Environmental exposures to tri-cresyl phosphates (TCPs) and the possible formation of toxic metabolites (e.g. cresyl saligenin phosphate; CBDP) may cause a variety of neurotoxic effects in humans. As reported for other organophosphorus compounds (OPs), the inhibition of acetylcholine esterase (AChE) has also been proposed as the underlying mechanism for TCP neurotoxicity. The ortho-isomer, ToCP and its metabolite CBDP are also known to affect neuropathy target esterase (NTE) leading to organophosphate-induced delayed neuropathy (OPIDN). Recently, in vitro testing has led to the identification of other molecular targets and alternative mechanisms of ToCP toxicity. The metabolite CBDP and other isomers, as well as commercial mixtures have not been tested for such additional modes of actions. Accordingly, the present study investigates alterations of neurobiological correlates of central nervous processes using different in vitro techniques. The three symmetric TCP isomers - ToCP, TpCP, and TmCP - that contain a methyl group at the ortho-, para-, or meta-position of the aromatic ring system, respectively, together with a commercial TCP mixture, and CBDP were all tested using concentrations not exceeding their cytotoxic concentrations. Isolated cortical neurons were kept in culture for 6days followed by 24h incubation with different concentrations of the test compounds. Thus, all endpoints were assessed after 7days in vitro (DIV 7), at which time cell viability, neurite microstructure, and the function of glutamate receptors and voltage-gated calcium cannels (VGCC) were measured. While the cytotoxic potential of the TCP isomers and their mixture were comparable (IC(50)>=80 M), CBDP was more cytotoxic (IC(50): 15 M) to primary cortical neurons. In contrast, CBDP (up to 10 M) did not compromise the microstructure of neurites. Ten M of ToCP significantly reduced the size and complexity of neurite networks, but neither TmCP and TpCP nor the mixture affected this second endpoint of neurotoxicity assessment. TCPs and their mixture significantly reduced the Ca(2+) influx in response to glutamate and KCl stimulation in concentrations of 10 M. Only ToCP showed a specific effect on glutamate receptors with 100nM reducing the evoked Ca(2+) influx. The effects of CBDP on the provoked Ca(2+) influx were much weaker than those observed for TCPs. These results confirmed that ToCP has a unique mode of action on glutamate receptors that are not observed with the metabolite CBDP and the other symmetric TCP isomers. In addition, the TmCP isomer seems to have the lowest potency with respect to inducing neurotoxic effects. CBDP did not affect the neurospecific endpoints investigated in this study. Therefore, the specific affinity of CBDP for NTE and the reported general cytotoxicity might be the most relevant modes of action of this toxic metabolite in the context of ToCP-induced neurotoxicity, including OPIDN.

Cresyl saligenin phosphate (CBDP) is a suspected causative agent of "aerotoxic syndrome", affecting pilots, crew members and passengers. CBDP is produced in vivo from ortho-containing isomers of tricresyl phosphate (TCP), a component of jet engine lubricants and hydraulic fluids. CBDP irreversibly inhibits butyrylcholinesterase (BChE) in human plasma by forming adducts on the active site serine (Ser-198). Inhibited BChE undergoes aging to release saligenin and o-cresol. The active site histidine (His-438) was hypothesized to abstract o-hydroxybenzyl moiety from the initial adduct on Ser-198. Our goal was to test this hypothesis. Mass spectral analysis of CBDP-inhibited BChE digested with Glu-C showed an o-hydroxybenzyl adduct (+106amu) on lysine 499, a residue far from the active site, but not on His-438. Nevertheless, the nitrogen of the imidazole ring of free l-histidine formed a variety of adducts upon reaction with CBDP, including the o-hydroxybenzyl adduct, suggesting that histidine-CBDP adducts may form on other proteins.

CSP (cresyl saligenin phosphate) is an irreversible inhibitor of human BChE (butyrylcholinesterase) that has been involved in the aerotoxic syndrome. Inhibition under pseudo-first-order conditions is biphasic, reflecting a slow equilibrium between two enzyme states E and E'. The elementary constants for CSP inhibition of wild-type BChE and D70G mutant were determined by studying the dependence of inhibition kinetics on viscosity and osmotic pressure. Glycerol and sucrose were used as viscosogens. Phosphorylation by CSP is sensitive to viscosity and is thus strongly diffusion-controlled (kon approximately 108 M-1.min-1). Bimolecular rate constants (ki) are about equal to kon values, making CSP one of the fastest inhibitors of BChE. Sucrose caused osmotic stress because it is excluded from the active-site gorge. This depleted the active-site gorge of water. Osmotic activation volumes, determined from the dependence of ki on osmotic pressure, showed that water in the gorge of the D70G mutant is more easily depleted than that in wild-type BChE. This demonstrates the importance of the peripheral site residue Asp70 in controlling the active-site gorge hydration. MD simulations provided new evidence for differences in the motion of water within the gorge of wild-type and D70G enzymes. The effect of viscosogens/osmolytes provided information on the slow equilibrium Eright harpoon over left harpoonE', indicating that alteration in hydration of a key catalytic residue shifts the equilibrium towards E'. MD simulations showed that glycerol molecules that substitute for water molecules in the enzyme active-site gorge induce a conformational change in the catalytic triad residue His438, leading to the less reactive form E'.

Mouse blood contains four esterases that detoxify organophosphorus compounds: carboxylesterase, butyrylcholinesterase, acetylcholinesterase, and paraoxonase-1. In contrast human blood contains the latter three enzymes but not carboxylesterase. Organophosphorus compound toxicity is due to inhibition of acetylcholinesterase. Symptoms of intoxication appear after approximately 50% of the acetylcholinesterase is inhibited. However, complete inhibition of carboxylesterase and butyrylcholinesterase has no known effect on an animal's well being. Paraoxonase hydrolyzes organophosphorus compounds and is not inhibited by them. Our goal was to determine the effect of plasma carboxylesterase deficiency on response to sublethal doses of 10 organophosphorus toxicants and one carbamate pesticide. Homozygous plasma carboxylesterase deficient ES1(-/-) mice and wild-type littermates were observed for toxic signs and changes in body temperature after treatment with a single sublethal dose of toxicant. Inhibition of plasma acetylcholinesterase, butyrylcholinesterase, and plasma carboxylesterase was measured. It was found that wild-type mice were protected from the toxicity of 12.5mg/kg parathion applied subcutaneously. However, both genotypes responded similarly to paraoxon, cresyl saligenin phosphate, diisopropylfluorophosphate, diazinon, dichlorvos, cyclosarin thiocholine, tabun thiocholine, and carbofuran. An unexpected result was the finding that transdermal application of chlorpyrifos at 100mg/kg and chlorpyrifos oxon at 14mg/kg was lethal to wild-type but not to ES1(-/-) mice, showing that with this organochlorine, the presence of carboxylesterase was harmful rather than protective. It was concluded that carboxylesterase in mouse plasma protects from high toxicity agents, but the amount of carboxylesterase in plasma is too low to protect from low toxicity compounds that require high doses to inhibit acetylcholinesterase.

CBDP [2-(2-cresyl)-4H-1-3-2-benzodioxaphosphorin-2-oxide] is a toxic organophosphorus compound. It is generated in vivo from tri-ortho-cresyl phosphate (TOCP), a component of jet engine oil and hydraulic fluids. Exposure to TOCP was proven to occur on board aircraft by finding CBDP-derived phospho-butyrylcholinesterase in the blood of passengers. Adducts on BChE, however, do not explain the toxicity of CBDP. Critical target proteins of CBDP are yet to be identified. Our goal was to facilitate the search for the critical targets of CBDP by determining the range of amino acid residues capable of reacting with CBDP and characterizing the types of adducts formed. We used human albumin as a model protein. Mass spectral analysis of the tryptic digest of CBDP-treated human albumin revealed adducts on His-67, His-146, His-242, His-247, His-338, Tyr-138, Tyr-140, Lys-199, Lys-351, Lys-414, Lys-432, and Lys-525. Adducts formed on tyrosine residues were different from those formed on histidines and lysines. Tyrosines were organophosphorylated by CBDP, while histidine and lysine residues were alkylated. This is the first report of an organophosphorus compound with both phosphorylating and alkylating properties. The o-hydroxybenzyl adduct on histidine is novel. The ability of CBDP to form stable adducts on histidine, tyrosine, and lysine allows one to consider new mechanisms of toxicity from TOCP exposure.

Aerotoxic syndrome is assumed to be caused by exposure to tricresyl phosphate (TCP), an antiwear additive in jet engine lubricants and hydraulic fluid. CBDP (2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one) is the toxic metabolite of triortho-cresylphosphate, a component of TCP. Human butyrylcholinesterase (BChE; EC 3.1.1.8) and human acetylcholinesterase (AChE; EC 3.1.1.7) are irreversibly inhibited by CBDP. The bimolecular rate constants of inhibition (k(i)), determined under pseudo-first-order conditions, displayed a biphasic time course of inhibition with k(i) of 1.6 x 10(8) M(-1) min(-1) and 2.7 x 10(7) M(-1) min(-1) for E and E' forms of BChE. The inhibition constants for AChE were 1 to 2 orders of magnitude slower than those for BChE. CBDP-phosphorylated cholinesterases are nonreactivatable due to ultra fast aging. Mass spectrometry analysis showed an initial BChE adduct with an added mass of 170 Da from cresylphosphate, followed by dealkylation to a structure with an added mass of 80 Da. Mass spectrometry in (18)O-water showed that (18)O was incorporated only during the final aging step to form phospho-serine as the final aged BChE adduct. The crystal structure of CBDP-inhibited BChE confirmed that the phosphate adduct is the ultimate aging product. CBDP is the first organophosphorus agent that leads to a fully dealkylated phospho-serine BChE adduct.

The restoration of plasma acetylcholinesterase activity in mice following inhibition by organophosphorus pesticides and nerve agents has been attributed to synthesis of new enzyme. It is generally assumed that activity levels return to normal, are stable and do not exceed the normal level. We have observed over the past 10 years that recovery of acetylcholinesterase activity levels in mice treated with organophosphorus agents (OP) exceeds pretreatment levels and remains elevated for up to 2 months. The most dramatic case was in mice treated with tri-cresyl phosphate and tri-ortho-cresyl phosphate, where plasma acetylcholinesterase activity rebounded to a level 250% higher than the pretreatment activity. The present report summarizes our observations on plasma acetylcholinesterase activity in mice treated with chlorpyrifos, chlorpyrifos oxon, diazinon, tri-ortho-cresyl phosphate, tri-cresyl phosphate, tabun thiocholine, parathion, dichlorvos, and diisopropylfluorophosphate. We have developed a hypothesis to explain the excess acetylcholinesterase activity, based on published observations. We hypothesize that acetylcholinesterase activity is induced when cells undergo apoptosis and that consequently there is a rise in the level of plasma acetylcholinesterase.

The aircraft cabin and flight deck ventilation are supplied from partially compressed unfiltered bleed air directly from the engine. Worn or defective engine seals can result in the release of engine oil into the cabin air supply. Aircrew and passengers have complained of illness following such "fume events". Adverse health effects are hypothesized to result from exposure to tricresyl phosphate mixed esters, a chemical added to jet engine oil and hydraulic fluid for its anti-wear properties. Our goal was to develop a laboratory test for exposure to tricresyl phosphate. The assay was based on the fact that the active-site serine of butyrylcholinesterase reacts with the active metabolite of tri-o-cresyl phosphate, cresyl saligenin phosphate, to make a stable phosphorylated adduct with an added mass of 80 Da. No other organophosphorus agent makes this adduct in vivo on butyrylcholinesterase. Blood samples from jet airplane passengers were obtained 24-48 h after completing a flight. Butyrylcholinesterase was partially purified from 25 ml serum or plasma, digested with pepsin, enriched for phosphorylated peptides by binding to titanium oxide, and analyzed by mass spectrometry. Of 12 jet airplane passengers tested, 6 were positive for exposure to tri-o-cresyl phosphate that is, they had detectable amounts of the phosphorylated peptide FGEpSAGAAS. The level of exposure was very low. No more than 0.05 to 3% of plasma butyrylcholinesterase was modified. None of the subjects had toxic symptoms. Four of the positive subjects were retested 3 to 7 months following their last airplane trip and were found to be negative for phosphorylated butyrylcholinesterase. In conclusion, this is the first report of an assay that detects exposure to tri-o-cresyl phosphate in jet airplane travelers.

Cytoskeletal components play an important role in maintaining cellular architecture and internal organization, with clear involvement of defining cell shape, in cell division and other cellular processes, such as neurite extension and maintenance. Alterations of cytoskeleton in human neuroblastoma SK-N-SH cells after exposure to different concentrations of tri-ocresyl phosphate (TOCP) for 12 hr were investigated. TOCP decreased the cell viability in a dose-dependent manner; the viability of SK-N-SH was reduced to approximately 50% of baseline after a 12-hour exposure to TOCP at high concentration (5 mM). Biochemical characterization by western blotting revealed that 1 and 5 mM concentrations of TOCP significantly inhibited the expression of neurofilament high molecular weight protein (NF-H), and that 5 mM TOCP inhibited expression of microtubule-associated protein 2c and tau protein, but not beta-actin. Indirect immunofluorescence analysis revealed that higher concentrations of TOCP decreased the length of neuritis and changed the structure of microfilaments, which are associated with NF-H. In addition, activities of neuropathy target esterase and acetylcholinesterase were significantly reduced after exposure to 5 mM TOCP for 12 hr. Together, these results suggested that the loss of cytoskeletal components is the early event during the process of TOCP toxicity towards human neuroblastoma SK-N-SH cells.

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.

Neurotoxicity of tricresyl phosphates (TCPs) and jet engine oil (JEO) containing TCPs were evaluated in studies conducted in both rat and hen. Results for currently produced samples ("conventional" and "low-toxicity") were compared with published findings on older samples to identify compositional changes and relate those changes to neurotoxic potential. Finally, a human risk assessment for exposure by oral ingestion of currently produced TCPs in JEO at 3% (JEO + 3%) was conducted. TCPs and certain other triaryl phosphates administered as single doses inhibited brain neuropathy target esterase (B-NTE; neurotoxic esterase) in the rat and the hen (hen 3.25 times as sensitive), and both species were deemed acceptable for initial screening purposes. Neither rat nor hen was sensitive enough to detect statistically significant inhibition of B-NTE after single doses of IEO + 3% "conventional" TCP. Subacute administration of 2 g/kg/d of JEO + 3% "conventional" TCP to the hen produced B-NTE inhibition (32%), which did not result in organophosphorus-induced delayed neurotoxicity (OPIDN). Subchronic administration of JEO + 3% TCP but not JEO + 1% TCP at 2 g/kg/d produced OPIDN. Thus, the threshold for OPIDN was between 20 and 60 mg "conventional" TCP/kg/d in JEO for 10 wk. The current "conventional" TCPs used in JEO and new "low-toxicity" TCPs now used in some JEO are synthesized from phenolic mixtures having reduced levels of ortho-cresol and ortho-xylenols resulting in TCPs of very high content of meta- and para-substituted phenyl moieties; this change in composition results in lower toxicity. The "conventional" TCPs still retain enough inhibitory activity to produce OPIDN, largely because of the presence of ortho-xylyl moieties; the "low-toxicity" TCPs are largely devoid of ortho substituents and have extremely low potential to cause OPIDN. The TCPs produced in the 1940s and 1950s were more than 400 times as toxic as the "low-toxicity" TCPs produced today. Analysis of the doses required to produce OPIDN in a subchronic hen study suggests that the minimum toxic dose of "conventional" TCP for producing OPIDN in a 70-kg person would be 280 mg/d, and for JEO containing 3% TCP, 9.4 g/d. Food products could be inadvertently contaminated with neat "conventional" TCP but it is unlikely that food such as cooking oil would be contaminated with enough JEO + 3% TCP to cause toxicity. Further, at the dosage required for neurotoxicity, it would be virtually impossible for a person to receive enough JEO + 3% TCP in the normal workplace (or in an aircraft) to cause such toxicity. There is no record of a JEO formulated with the modern "conventional" TCP causing human neurotoxicity.

Carboxylesterases (CbxE) can be inhibited by organophosphorus esters (OPs) without causing clinical evidence of toxicity. CbxE are thought to protect the critical enzyme acetylcholinesterase (AChE) from OP inhibition in animals. CbxE and AChE are both present in neuroblastoma cells, but, even though these cells have potential to be an in vitro model of OP toxicity, the effect of OPs on CbxE and the relationship of CbxE inhibition and AChE inhibition have not yet been examined in these cells. Therefore, this study examined concentration-related OP-induced inhibition of CbxE in human SH-SY5Y and mouse NB41A3 neuroblastoma cells with 11 active esterase inhibitors: paraoxon, malaoxon, chlorpyrifos-oxon, tolyl saligenin phosphate (TSP), phenyl saligenin phosphate (PSP), diisopropyl phosphorofluoridate (DFP), mipafox, dichlorvos, trichlorfon, dibutyryl dichlorovinyl phosphate (DBVP), and dioctyl dichlorovinyl phosphate (DOVP). All could inhibit CbxE, although the enzyme was less likely to be inhibited than AChE following exposure to 9 of the test compounds in the human cell line and to all 11 of the test compounds in the murine cell line. Species differences in concentration-related inhibitions of CbxE were evident. When cells were exposed first to an OP with a low IC50 toward CbxE (PSP), followed by an OP with high affinity for AChE (paraoxon or malaoxon), inhibitions of CbxE and AChE were additive. This indicated that CbxE did not protect AChE from OP-induced inhibition in this cell culture model.

This study reports the activity of neurotoxic esterase (NTE) and acetylcholinesterase (AChE) in the blood and central nervous system (CNS) of swine 6, 12, 24 and 48 h after a single oral dose of 800 mg tri-o-cresyl phosphate (TOCP)/kg. At all evaluated intervals, inhibition of NTE activity in leukocytes and the CNS was 88% or higher, with only slight differences in NTE inhibition apparent among the various tissues examined. This extreme inhibition of NTE activity precluded correlation between inhibition of NTE in peripheral leukocytes and the CNS. However, the similarity in NTE response in leukocytes and the CNS following TOCP administration indicates the potential for leukocyte NTE as a biochemical marker for organophosphorus ester-induced delayed neurotoxicity (OPIDN) development. As the distribution pattern of NTE in the CNS of swine closely parallels that of man, these results further suggest that swine may prove a useful animal model for the study of OPIDN. The activity of AChE was highly variable based on time of assay and tissue examined. In general, plasma AChE activity was more severely depressed in all animals and responded more rapidly to TOCP administration. However, erythrocyte AChE more accurately reflected the enzyme's activity in the CNS and the clinical response to TOCP. Based on the data provided by this study, a threshold inhibition of erythrocyte AChE between 59 and 74% is required for production of acute cholinergic signs.

We report the clinical signs and the effects on leukocyte neurotoxic esterase and red blood cell and plasma acetylcholinesterase (AChE) activities in swine orally administered a single dose of tri-o-cresyl phosphate (TOCP) at 400, 800 or 1000 mg/kg. Swine in all dosage groups exhibited signs consistent with inhibition of nervous tissue cholinesterase 3-48 h after TOCP administration. Onset was dose-related, and 2/3 1000 mg/kg dosed swine died 3 or 35 h postdosing. In surviving swine, significant depressions in plasma AChE activity were apparent at 6 h postdosing, ranging from 16-23% of predosing levels. Similar depressions of red blood cell AChE were not observed until 24 h postdosing. Plasma AChE activities appeared to more accurately reflect the development of acute cholinergic signs observed in the 1000 mg/kg dosed swine at 3 h postdosing while red blood cell AChE activities were more consistent with the delayed cholinergic signs exhibited by the 400 and 800 mg/kg dosed swine at 24 h postdosing. All survivors developed signs of delayed neurotoxicity 10-12 d after TOCP administration, and 70% or greater inhibition of neurotoxic esterase activity in leukocytes was apparent during the first 48 h postdosing.

Six trace elements were monitored in neural tissue homogenates from White Leghorn hens orally dosed with tri-o-tolyl phosphate (TOTP) or tri-m-tolyl phosphate (TMTP) (200 mg/kg). Treated birds were monitored daily for development of delayed neurotoxicity, and concentrations of calcium, copper, iron, magnesium, manganese, and zinc were measured with atomic absorption spectroscopy at the time of maximal locomotor impairment (27-35 days postdosing). TOTP-treated birds manifested motor deficit by 15 days postdosing, while hens administered TMTP exhibited no signs of delayed neurotoxicity. Total calcium content in the sciatic nerve homogenates from TOTP-dosed hens was significantly less (P < 0.05) at the time of maximal locomotor impairment, while no shifts in the other trace elements were found. Therefore, the ortho isomer of tritolylphosphate elicited symptoms of delayed neurotoxicity in the hen (i.e., organophosphorus ester-induced delayed neurotoxicity or OPIDN) and caused a decrease in total calcium content in the sciatic nerve homogenates, in contrast to effects of the meta isomer. Analysis of neural homogenates at time of maximal locomotor impairment reflected secondary events in the degradative processes, since the initial assault of TOTP happens early after administration. Therefore, at fully developed OPIDN alteration of calcium balance in sciatic nerves is an indicator of axonopathy in a degenerated nerve following chemical injury.

The in vivo effect of a single dose of the neuropathic compound triorthocresyl-phosphate (TOCP) on phosphofructokinase (PFC, E.C. 2.7.1.11) and its relation with the initiation step (inhibition and aging of neuropathy target esterase, NTE) in the TOCP-induced delayed neuropathy have been studied. Hens were treated with a neurotoxic dose of TOCP (500 mg/kg, p.o.) and with a protective compound (Phenylmethanesulfonyl fluoride, PMSF, 30 mg/kg s.c.) in different combinations: TOCP, TOCP + PMSF, PMSF + TOCP and PMSF. PFK activity was determined in brain and sciatic nerve 1, 3, 7 and 15 days after treatment. PFK activity decreased in sciatic nerve 15 days after dosing with TOCP or TOCP + PMSF. When animals were dosed with the protective agent (PMSF) alone or before administering the neurotoxic compound, PFK activity was unaltered and clinical signs of neuropathy were absent. The data presented here suggest that phosphofructokinase is involved in the pathogenesis of the neuropathy induced by TOCP.

Previous studies have shown that after dosing with tri-o-cresyl phosphate (TOCP), the testis contains more active intermediate (saligenin cyclic-o-tolyl phosphate; SCOTP) than do other organs or blood. SCOTP is produced by a cytochrome P450-dependent reaction, and the Sertoli cells, although containing little P450, are the testicular cells that show the first signs of damage after TOCP administration. The present studies evaluated (i) whether testicular Leydig cell production of SCOTP might explain the elevated testicular concentration of SCOTP, (ii) if this production affected testosterone secretion, and (iii) if Sertoli cells cocultured over TOCP-exposed Leydig cells would show effects similar to those found after SCOTP exposure of Sertoli cells in vitro, indicating a cell interaction. Previous data showed that a target enzyme for SCOTP in Sertoli cells, nonspecific esterase (NSE), was inhibited by exposure in vitro to SCOTP, but not to TOCP. In the present experiments, HPLC analysis identified SCOTP in media from Leydig cells cultured with radiolabeled TOCP, demonstrating activation. TOCP addition to Leydig cells decreased testosterone output after stimulation with hCG, an effect that was replicated by subsequent in vivo experiments. Addition of various intermediates in the testosterone biosynthesis pathway indicated that both mitochondrial- and microsomal-based steps in the pathway were affected. Collectively, these data indicate that Leydig cells can activate TOCP. To model whether this activation might affect Sertoli cells in vivo, Sertoli cells were plated in culture-well inserts suspended above (cocultured with) isolated Leydig cells in the presence of TOCP. Sertoli NSE activity was diminished, while remaining unchanged when cultured in the presence of TOCP but without Leydig cells, or over Leydig cells alone. These results show that the Leydig cells in the testis are capable of activating TOCP to SCOTP, and that this can produce effects in Sertoli cells. This in situ activation of TOCP to SCOTP may help explain why the testis contains high concentrations of SCOTP after in vivo dosing with TOCP, and why the testis is a target organ for TOCP toxicity.

The activity of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNP, EC 3.1.4.37), a myelin-associated enzyme, was monitored in brain, spinal cord, and sciatic nerve homogenates from tri-o-tolyl phosphate (TOTP) and tri-m-tolyl phosphate (TMTP) treated hens. Atropinized adult White Leghorn hens were orally dosed with TOTP (200 mg/kg) or with TMTP (200 mg/kg). The treated birds were monitored daily for development of delayed neurotoxicity, and CNP activity was measured via spectrophotometry at the time of maximal locomotor impairment (27-35 days post dosing). The TOTP-treated birds manifested locomotor deficit by 15 days postdosing and exhibited T2-T4 ataxia at maximal locomotor impairment. The hens administered TMTP exhibited no signs of delayed neurotoxicity. CNP activity of sciatic nerve preparations from TOTP-dosed hens was significantly inhibited (p less than 0.05) at the time of maximal locomotor impairment. There was also a significant correlation between decreased CNP activity and the degree of ataxia at the time of maximal locomotor impairment. This decrease in sciatic nerve CNP activity was most likely associated with nerve fiber degeneration. The level of CNP activity in spinal cord and brain homogenates from TOTP-dosed birds was not markedly altered. TMTP-treated birds exhibited no change in neural tissue CNP activity. The results suggest that the criterion of decreased CNP activity may serve as a useful biochemical adjunct to established clinical, biochemical, and morphological methods in the assessment of chemically-induced neuropathies.

The poor absorption of organophosphate delayed neurotoxins through the gastrointestinal tract has been suggested as a reason why young chickens are not susceptible to organophosphate-induced delayed neurotoxicity (OPIDN). In the present study, 4-wk-old White Leghorn chickens were administered a single dose of 500 mg tri-o-tolyl phosphate (TOTP)/kg body weight or 100 mg o-tolyl saligenin phosphate (TSP)/kg body weight via the oral, intramuscular, or intraperitoneal route. In addition, TOTP TSP were administered intravenously at 250 and 50 mg/kg body weight, respectively. Forty-eight hours after dosing, half the birds in each group were killed for subsequent determination of whole-brain and sciatic nerve neurotoxic esterase (NTE) activity while the remaining 5 birds per group were observed daily from d 7 through d 21 for development of OPIDN clinical signs. TOTP administered by the 4 routes generally resulted in whole-brain and sciatic nerve NTE inhibition in excess of 85%. TSP given via the different routes resulted in 75-84% inhibition of whole-brain NTE activity and 66-79% inhibition of sciatic nerve NTE activity. No birds displayed clinical signs typical of OPIDN during the 21-d test. Thus, the resistance of the young chicken to the delayed effects of organophosphate compounds is due to factors other than the poor absorption of the compound through the gastrointestinal tract or the inability of the bird to convert TOTP to its neuroactive metabolite, TSP.

Simultaneous intoxication with hexacarbon solvents and organophosphorus compounds has been considered a possible critical factor in some occupational neuropathies and their interactions proved to cause potentiation effects in hens [1-3]. A high degree of inhibition of neuropathy target esterase (NTE) is needed to develop organophosphorus induced polyneuropathy (OPIDP). In this work, the inhibition of NTE, BCHE and AChE by TOCP on control and n-hexane pretreated (7-15 days, 300 mg/kg per day) hens is studied. Using a single TOCP dose of 200 mg/kg, n-hexane pretreated hens showed synergistic effects, but no significant differences were observed in the inhibition of cholinesterases and NTE in brain or spinal cord. With lower TOCP dose (20 mg/kg) statistically significant differences were observed, which were not drastic but could be important because they involved an increase of inhibition up to critical threshold values (from 40-50% to 60-70% inhibition). However, no clinical effects were observed in these animals. Possible mechanisms of neurotoxic interaction are discussed.

The neurotoxicities of single doses of a chemical warfare agent VX [phosphonothioic acid, methyl-S-(2-[bis(1-methylethyl)amino/ethyl) O-ethyl ester], a metabolite of the agricultural chemical parathion, paraoxon, PO (phosphonothioic acid, diethyl paranitrophenyl ester), and the known neuropathic agents DFP] phosphorofluoridic acid, bis(1-methylethyl) ester] and TOCP (phosphoric acid, tri-o-tolyl ester) were compared in the chicken. Single injections (subcutaneous, sc) of VX as high as 150 micrograms/kg (5 times the LD50, intramuscular, im) were tolerated by laying tens if atropine and 2-pralidoxime were used as antidotes before and immediately after injection. The 150 of VX for inhibition of chicken brain acetylcholinesterase was approximately 5 X 10(-10). Plasma acetylcholinesterase, but not butyrylcholinesterase, was depressed 2 h after injections of 2-20 micrograms VX/kg im without antidotes. Levels of plasma enzymes such as creatine kinase, indicative of tissue damage, were increased after exposure to both VX and PO. Injections of up to 150 micrograms/kg of VX with antidotes did not cause locomotor or histological signs of organophosphorus-induced delayed neuropathy, but single injections of 400 mg TOCP/kg did.

Hens were given a single oral dose (0.235 mg/kg) of tri-o-cresylphosphate (TOCP) during chronic n-hexane treatment (200 mg/kg daily, 5 days/week). They were compared with other animals treated only with n-hexane or only with TOCP. Animals treated with a higher TOCP dose (1 ml/kg) were used as positive controls. The animals treated with both n-hexane and TOCP showed rapid development of severe ataxia. The rate of the ataxia development was similar to that of the positive controls but with earlier onset of the first signs and with less loss of body weight. However, animals treated only with n-hexane, under the same conditions, showed only reversible weakness and sedative effects, and those treated only with TOCP showed slow and slight ataxia development. The n-hexane- and TOCP-treated hens showed axonal swelling with myelin retraction associated with Ranvier's nodes, which is characteristic of long hexacarbon exposure. Some internodal swellings were also observed but less frequently than the paranodal swellings. The time course of the ataxia development was similar to an organophosphorus-induced delayed neuropathy (OPIDN); however, the light microscopy observation more closely resembled hexacarbon neuropathy. The results suggest a potentiation effect of n-hexane and TOCP neurotoxicities which could be related to some human occupational neuropathies.

A commercial cresyl diphenyl phosphate preparation was analyzed to contain approximately 35% of triphenyl phosphate, 45% of cresyl diphenyl phosphates, 18% of dicresyl phenyl phosphates and 2% of tricresyl phosphates. The product was almost free of the o-cresyl isomers as revealed by the analysis of its alkaline hydrolysis products. A single intraperitoneal injection (150 or 300 mg/kg) caused an induction of microsomal cytochrome P-450 in the liver of Wistar rats with a concomitant increase in the activities of mixed function monooxygenases and proliferation of smooth endoplasmic reticulum 24 h after the treatment. These effects were not detected in the kidneys. The morphological changes in hepatocytes included the enlargement of nuclei and mitochondria with increased cristae. The hepatic morphology returned to normal 2 weeks after the treatment. The activity of pseudocholine esterase in blood was inhibited 4 h and 24 h after the injection but the effect levelled off. The concentration of the organophosphates in blood and liver decreased rapidly with only traces detected in blood after 24 h. No effects on the activities of cerebral and muscle acetylcholine esterase were observed. The treatment (300 mg/kg) inhibited the brain--2',3'-cyclic nucleotide 3'-phosphohydrolase through the 2-week observation period associated with demyelination in peripheral nerves.

The sensitivity of the mouse to organophosphorus-induced delayed neurotoxicity (OPIDN) has been investigated. One group of five mice received two single 1000-mg/kg po doses of tri-o-cresyl phosphate (TOCP) at a 21-day interval (on Days 1 and 21 of the study); a second group of five mice was given 225 mg/kg of TOCP daily for 270 days. A third group of five animals served as an untreated control. All animals were killed 270 days after the start of the experiment. Daily po dosing of 225 mg/kg TOCP caused a decrease in body weight gain, muscle wasting, weakness, and ataxia which progressed to severe hindlimb paralysis at termination. On the other hand, po administration of two single 1000-mg/kg doses of TOCP at a 21-day interval produced no observable adverse effects. Brain acetylcholinesterase (AChE) and neurotoxic esterase (NTE) activity were 35 and 10% of the control, respectively, in daily dosed animals while AChE and NTE in mice receiving two single 1000-mg/kg doses of TOCP were not significantly altered from the control group. Plasma butyrylcholinesterase activity was 12% of the control group in daily dosed animals. Hepatic microsomal enzyme activities of aniline hydroxylase and p-chloro-N-methylaniline demethylase and NADPH-cytochrome P-450 content in daily dosed animals were increased (141 to 161% of the control group) when compared to controls and mice receiving two single 1000-mg/kg doses of TOCP; the latter being not significantly different from each other. Degeneration of the axon and myelin of the spinal cord and sciatic fascicle were observed and were consistent with OPIDN. This study demonstrates that chronic dosing of TOCP produces OPIDN and induces hepatic microsomal enzyme activity in mice. It is concluded that while the mouse is susceptible to OPIDN, it is a less sensitive and a less appropriate test animal for studying this effect when compared to the adult hen.

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.

Nine esterase fractions hydrolyzing 1-naphthylacetate were revealed in Triton X-100-solubilized extracts from aphides homogenates by polyacrylamide gel electrophoresis. The less mobile fractions 1-4 were identified as cholinesterases, using specific inhibitors--eserine and the cationic phosphoorganic inhibitor Gd-42; fractions 5-7 were related to carboxylesterases, using specific inhibition by triorthocresylphosphate and O,O-dimethyl (2,2-dichlorvinyl)phosphate. The most mobile fractions 8-9 which were resistant to the inhibitors, were classified as arylesterases. The aphis cholinesterase fractions revealed the highest mobility; the activity of carboxylesterase fractions was lower. Thiophosphonate--C8H17O(CH3)P(O)-SCH2SCH2COOCH3 was found to be a highly efficient selective inhibitor of aphis carboxylesterase, i. e. the kII values for carboxylesterase and cholinesterase were equal to 10(8) and 10(5) M-1 min-1, respectively. The thiophosphoorganic derivatives containing a beta-alanine residue in the cleaved part are more specific to acetylcholinesterase and carboxylesterase than those containing a valine residue. Studies with enanthiomers--C2H5O(CH3)P(O)SCH2CONHCH2CH2COOC2H5 and (C2H5O)2P(O)SCH2CONHCH(iC3H7)COOC2H5 have demonstrated that the asymmetry due to the central phosphorus atom is more essential for the acetylcholinesterase and carboxylesterase activities than that connected with the carbon atom in the cleaved part of the inhibitor molecule. During the interaction of the enanthiomers with the asymmetric phosphorus the stereospecificity of acetylcholinesterase is much higher than that of carboxylesterase. In terms of stereospecificity of the esterase site aphis acetylcholinesterase is is similar to its mammalian counterpart, while carboxylesterase from the same source is rather close to mammalian butyrylcholinesterase.

Tri-ortho-cresyl phosphate (TOCP) was given orally or by subcutaneous (SC) injection to sheep and swine. Sheep given oral doses of 100, 200, or 400 mg of TOCP/kg of body weight developed an acute intoxication characterized by diarrhea dehydration, metabolic acidosis, and death within 6 days. Daily SC injections of TOCP in sheep caused either death or delayed neurotoxicosis depending upon the dosage. Increase of aspartate aminotransferase activity approximately 24 hours before the animal died and histopathologic changes confirmed that liver injury had occurred. Swine dosed with 100 to 1,600 mg of TOCP/kg had minimal signs of acute toxicosis, but developed severe delayed neurotoxicosis in approximately 15 days. Those given a 800 mg/kg dose by the oral route or SC injection had severely decreased serum acetylcholinesterase activity. In the swine which were euthanatized at 7 days after treatment, histopathologic examinations revealed no lesions (although the nervous system was not examined, because clinical neurologic signs were normal).

The aim of the experiments was to obtain more information on the toxicity of organophosphates and protection against them. Pretreatment of mice with CBDP increased the s.c. toxicity of soman 19.1-fold, and its i.p. toxicity 17.8-fold. The protective effect of atropine and the oximes HS-3, HS-6 and HI-6 in soman poisoning was much greater in CBDP pretreated than in control animals. Atropine + HI-6 raised the s.c. LD50 of soman in the CBDP pretreated animals from 6.8 micrograms/kg to 166 micrograms/kg (PI = 24.3), but in control animals the i.p. LD50 was only raised from 370 micrograms/kg to 608 micrograms/kg (PI = 0.6). CBDP inhibited blood and brain AChE activity, but had no effect on aliesterase (AE) activity in plasma, liver and brain of mice in vivo. CBDP increased s.d. toxicities of sarin 11-fold, of tabun 5-fold and of VX 0.24-fold. The protective index of atropine + HS-3 in sarin poisoning, as in the case of soman poisoning, was much higher in CBDP pretreated than in control animals (20.1 : 13.6), only slightly higher in tabun poisoning (4.3 : 3.4) and in the case of VX poisoning lower in CBDP pretreated than in control animals (32 : 47). The results indicate that CBDP potentiates soman, sarin and tabun toxicities mainly by blocking their binding to non-specific sites in the body.

The effect of tricresylphosphate (TCP) was studied in vitro and in vivo on the rat liver and brain enzymes acetylcholinesterase (ACC), butyrylcholinesterase (CHE), arylesterase (ARE), aliesterase (ALI), and the microsomal nicotinamide-adenine dinucleotide phosphate oxidase (NADPH2-oxidase) system. The results show that, in the male rat, TCP given intraperitoneally induces an increase in liver microsomal ARE AND NADPH2-oxidase and a decrease in ALI and cholinesterase; no activation of ARE and NADPH2-oxidase is observed in female rats.

Ten day old chick sympathetic ganglia cultured in a microslide assembly were treated with a selected group of organophosphate pesticides to evaluate their cytotoxicity ranges, and the usefulness of such a model for screening pesticides. Examination by phase contrast and light microscopy for chemically-induced morphological alteration of nerve fibers, glial cells and neurons provided the criteria for quantitation and assessment of the toxic effects. Concentrations that produced half-maximal effects ranged from 1 x 10(-6)M (severely toxic) for methylparathian, diazinon, paraoxon, mevinphos, diisopropylfluorophosphate, tri-o-tolyl phosphate and its mixed isomers to a 1 x 10(-3)M (intermediate) for malathion, leptophos, coumaphos, mono- and dicrotophos. Some or no effects were evident at 1 x 10(2-)M for O'ethyl-O-p-nitrophenyl phenyl phosphonothioate, tri-m-tolylphosphate, chlorpyriphos and triphenyl phosphate. In all instances, nerve fibers were more sensitive than neurons or glial cells to insecticides. All cellular growth was inhibited at 1 x 10(-2)M (except triphenyl phosphate). Below 1 x 10(-7)M, no inhibitory effects were evident. The secondary abnormalities included decreased cellular migration, diffuse cellular growth pattern, increased vacuolization, nerve fiber swelling and cellular degeneration. The cytotoxic effects of these chemicals do not appear to be related to in vivo toxicity or cholinesterase inhibition potential.

Chicken spinal cord adenosine triphosphatases (both Na+, K+ stimulated and ouabain insensitive) were inhibited by tri-o-tolyl phosphate (TOTP, a neurotoxic organophosphate which is not a cholinesterase inhibitor) and mevinphos (a non-neurotoxic compound but inhibitor of cholinesterases). The inhibition was concentration and time dependent, with an initial rapid drop in activity followed by a gradual exponential decline.

TRI-O-CRESYL PHOSPHATE (TOCP) is metabolized in vitro and in vivo to form potent esterase inhibitors15. The nature and biological activity of the metabolites were investigated"

Tri-p-ethylphenyl phosphate is unique amongst the organophosphorus compounds which produce neurotoxic effects in not being an inhibitor of cholinesterase. The dysfunction it produces is also marked by some unusual features. Thus it produces a characteristic high-stepping gait which develops at varying periods after intramuscular injection but more regularly following oral administration. A careful comparison of the character, onset and development of the effects of diisopropyl phosphorofluoridate (dyflos), tri-o-cresyl phosphate and tri-p-ethylphenyl phosphate has illustrated the differences between the two former substances and tri-p-ethylphenyl phosphate. It has also confirmed a previous suggestion that this substance acts in a different manner from the other two, a suggestion supported by the histological evidence.