Inhibitor Report for: CBDP

The dose-response (0.1 to 1000 mg/kg sc) effects of 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide (CBDP; a metabolite of the organophosphorus compound tri-o-cresylphosphate) on total cholinesterase (ChE) and carboxylesterase (CaE) activities in tissues from the rat were examined. Doses of CBDP greater than 1.0 mg/kg inhibited CaE activity maximally (greater than 99%) in plasma and lung, two important sites for detoxification of organophosphorus toxicants. A biphasic dose-dependent inhibition of ChE activity was seen in all tissues; the ED50 values showed a difference of two orders of magnitude between the first and the second phases of the dose-response curves. CBDP inhibited the blood esterases in the order plasma CaE much greater than plasma ChE much greater than red blood cell (RBC) ChE. The biphasic dose-response curve and preferential inhibition of the blood esterases may reflect the inhibition of butyrylcholinesterase in preference to acetylcholinesterase in these tissues. At doses of CBDP below 1.0 mg/kg, plasma, RBC, and brain regional ChE activities were inhibited by less than 10%, whereas at doses above 2.0 mg/kg, ChE activities were inhibited substantially (up to 80% in plasma, up to 60% in RBC, and greater than 90% in brain regions). On the basis of these results, a dose of CBDP between 1.0 and 2.0 mg/kg should prove useful as a pretreatment for studies of OP toxicity in the rat.

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.

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"

Kinetic studies and molecular modeling of human acetylcholinesterase (AChE) inhibition by a fluorinated acetophenone derivative, 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (TFK), were performed. Fast reversible inhibition of AChE by TFK is of competitive type with K(i) = 5.15 nM. However, steady state of inhibition is reached slowly. Kinetic analysis showed that TFK is a slow-binding inhibitor (SBI) of type B with K(i)* = 0.53 nM. Reversible binding of TFK provides a long residence time, = 20 min, on AChE. After binding, TFK acylates the active serine, forming an hemiketal. Then, disruption of hemiketal (deacylation) is slow. AChE recovers full activity in approximately 40 min. Molecular docking and MD simulations depicted the different steps. It was shown that TFK binds first to the peripheral anionic site. Then, subsequent slow induced-fit step enlarged the gorge, allowing tight adjustment into the catalytic active site. Modeling of interactions between TFK and AChE active site by QM/MM showed that the "isomerization" step of enzyme-inhibitor complex leads to a complex similar to substrate tetrahedral intermediate, a so-called "transition state analog", followed by a labile covalent intermediate. SBIs of AChE show prolonged pharmacological efficacy. Thus, this fluoroalkylketone intended for neuroimaging, could be of interest in palliative therapy of Alzheimer's disease and protection of central AChE against organophosphorus compounds.

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.

Tri-ortho-cresyl phosphate (TOCP) has been widely used as plasticizers, plastic softeners, and flame-retardants in industry, which can be metabolized to High-toxic saligenin cyclic-o-tolyl phosphate (SCOTP). Our previous results found that TOCP could disrupt the seminiferous epithelium in the testis and induce autophagy of rat spermatogonial stem cells. Little is known about the toxic effect of SCOTP on rat spermatogonial stem cells. The present study showed that SCOTP decreased viability of rat spermatogonial stem cells in a dose-dependent manner. Both LC3-II and the ratio of LC3-II/LC3-I were significantly increased; autophagy proteins atg5 and Beclin 1 were also markedly increased after treatment with SCOTP, indicating SCOTP could induce autophagy of the cells. Ultrastructural observation under the transmission electron microscopy (TEM) indicated that there were autophagic vacuoles in the cytoplasm in the SCOTP-treated cells. However, cell cycle arrest was not observed by flow cytometry; and the mRNA levels of p21, p27, p53 and cyclin D1 in the cells were also not affected by SCOTP. Meanwhile, SCOTP didn't induce apoptosis of the cells. In summary, we showed that SCOTP could induce autophagy of rat spermatogonial stem cells, without affecting cell cycle and apoptosis.

Tri-o-cresyl-phosphate (TOCP) is a common additive in jet engine lubricants and hydraulic fluids suspected to have a role in aerotoxic syndrome in humans. TOCP is metabolized to cresyl saligenin phosphate (CBDP), a potent irreversible inhibitor of butyrylcholinesterase (BChE), a natural bioscavenger present in the bloodstream, and acetylcholinesterase (AChE), the off-switch at cholinergic synapses. Mechanistic details of cholinesterase (ChE) inhibition have, however, remained elusive. Also, the inhibition of AChE by CBDP is unexpected, from a structural standpoint, i.e., considering the narrowness of AChE active site and the bulkiness of CBDP. In the following, we report on kinetic X-ray crystallography experiments that provided 2.7-3.3 A snapshots of the reaction of CBDP with mouse AChE and human BChE. The series of crystallographic snapshots reveals that AChE and BChE react with the opposite enantiomers and that an induced-fit rearrangement of Phe297 enlarges the active site of AChE upon CBDP binding. Mass spectrometry analysis of aging in either H(2)(16)O or H(2)(18)O furthermore allowed us to identify the inhibition steps, in which water molecules are involved, thus providing insights into the mechanistic details of inhibition. X-ray crystallography and mass spectrometry show the formation of an aged end product formed in both AChE and BChE that cannot be reactivated by current oxime-based therapeutics. Our study thus shows that only prophylactic and symptomatic treatments are viable to counter the inhibition of AChE and BChE by CBDP.

Poisoning by organophosphate nerve agents can induce seizures which rapidly become refractory to treatment and result in brain damage. Current therapies have only a narrow time frame for effective administration after poisoning. 5-HT1A agonists were tested for efficacy in mice against a seizure-producing combination of the carboxylesterase inhibitor 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide (CBDP) and sarin, producing an LD20-40. Administration of the 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) decreased glial fibrillary acidic protein (GFAP) staining in mice when administered 1min after CBDP and sarin while other 5-HT1A agonists buspirone and S-14506 were not effective. The reduction in GFAP staining by 8-OH-DPAT remained significant when a single dose was administered 2h after the toxic challenge. In addition, 8-OH-DPAT reversed the increase in the inflammatory factor IL-1beta in the dentate gyrus and amygdala but did not reduce positive TUNEL staining in the dentate gyrus. Due to the failure of the two other agonists to provide protection, the 5-HT1A antagonist WAY-100635 was tested. WAY-100635 was found to neither reverse the neuroprotective effects of 8-OH-DPAT nor worsen the damage when given alone, making a role for this receptor unlikely. The neuroprotective effects of 8-OH-DPAT appear to lie within its secondary pharmacology.

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'.

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.

Sub-lethal exposure to sarin (GB), a potent chemical warfare agent, produces long-term neurological deficits in both humans and rodents. However, rodents express much higher levels of carboxylesterase (CaE) than humans and require a much higher dose of GB in rodents to produce neurotoxicity. In mice, the combination of the carboxylesterase inhibitor 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide (CBDP) with the organophosphorus (OP) nerve agent GB renders mice more sensitive to OP poisoning. After the reduction in CaE, GB inhibits acetylcholinesterase at doses similar to those in human toxicity. A dose-response curve for GB was determined in male C57BL/6 mice after 1.5mg/kg CBDP. A functional observational battery (FOB) for behavior was used to determine the dose needed to elicit seizure activity but maintain a mortality of less than 50%. Neuronal cell death was evaluated at 4, 7, 10 and 14 days post-GB exposure. Multiple brain areas were examined using cresyl violet: CA1 and the dentate gyrus of the hippocampus, amygdala and piriform cortex. GFAP staining was then measured as an index of cell death in the dentate gyrus of the hippocampus. The dentate gyrus and CA1 exhibited significant neuronal death indicated by both cresyl violet and GFAP staining. The treated animals also had a significant decrease in tissue and blood acetylcholinesterase, in addition to decreases in plasma CaE. CBDP renders mice more sensitive to the effects of GB exposure and mirrors a human symptomatic exposure dose.

Aerotoxic syndrome is assumed to be caused by exposure to tricresyl phosphate, an additive in engine lubricants and hydraulic fluids that is activated to the toxic 2-(ortho-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP). Currently, there is no laboratory evidence to support intoxication of airline crew members by CBDP. Our goal was to develop methods for testing in vivo exposure by identifying and characterizing biomarkers. Mass spectrometry was used to study the reaction of CBDP with human albumin, free tyrosine, and human butyrylcholinesterase. Human albumin made a covalent bond with CBDP, adding a mass of 170amu to Tyr411 to yield the o-cresyl phosphotyrosine derivative. Human butyrylcholinesterase made a covalent bond with CBDP on Ser198 to yield five adducts with added masses of 80, 108, 156, 170, and 186amu. The most abundant adduct had an added mass of 80amu from phosphate (HPO(3)), a surprising result given that no pesticide or nerve agent is known to yield phosphorylated serine with an added mass of 80amu. The next most abundant adduct had an added mass of 170amu to form o-cresyl phosphoserine. It is concluded that toxic gases or oil mists in cabin air may form adducts on plasma butyrylcholinesterase and albumin, detectable by mass spectrometry.

Comparative protection studies in mice demonstrate that on a molar basis, recombinant human acetylcholinesterase (rHuAChE) confers higher levels of protection than native human butyrylcholinesterase (HuBChE) against organophosphate (OP) compound intoxication. For example, mice challenged with 2.5 LD50 of O-isopropyl methylphosphonofluoridate (sarin), pinacolylmethyl phosphonofluoridate (soman), and O-ethyl-S-(2-isopropylaminoethyl) methylphosphonothiolate (VX) after treatment with equimolar amounts of the two cholinesterases displayed 80, 100, and 100% survival, respectively, when pre-treatment was carried out with rHuAChE and 0, 20, and 60% survival, respectively, when pretreatment was carried out with HuBChE. Kinetic studies and active site titration analyses of the tested OP compounds with acetylcholinesterases (AChEs) and butyrylcholinesterases (BChEs) from different mammalian species demonstrate that the superior in vivo efficacy of acetyl-cholinesterases is in accordance with the higher stereoselectivity of AChE versus BChE toward the toxic enantiomers comprising the racemic mixtures of the various OP agents. In addition, we show that polyethylene glycol-conjugated (PEGy-lated) rHuAChE, which is characterized by a significantly extended circulatory residence both in mice and monkeys ( Biochem J 357: 795-802, 2001 ; Biochem J 378: 117-128, 2004 ), retains full reactivity toward OP compounds both in vitro and in vivo and provides a higher level of protection to mice against OP poisoning, compared with native serum-derived HuBChE. Indeed, PEGylated rHuAChE also confers superior prophylactic protection when administered intravenously or intramuscularly over 20 h before exposure of mice to a lethal dose of VX (1.3-1.5 LD50). These findings together with the observations that the PEGylated rHuAChE exhibits unaltered biodistribution and high bioavailability present a case for using PEGylated rHuAChE as a very efficacious bioscavenger of OP agents.

Earlier, we have shown that rat hepatic and pancreatic fatty acid ethyl ester (FAEE) synthases are structurally and functionally similar to rat liver carboxylesterase (CE) and pancreatic cholesterol esterase (ChE), respectively. We have also reported that only hepatic FAEE synthase is inhibited by tri-o-tolylphosphate (TOTP) in vivo and in human hepatocellular carcinoma (HepG2) cells. The metabolism of TOTP is a prerequisite for the inhibition of hepatic FAEE synthase as well as esterase activity. To further elucidate the mechanism of such differential inhibition by inhibitors of serine esterases, we synthesized two metabolites of TOTP, 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphoran-2-one (CBDP; cyclic saligenin phosphate) and di-o-tolyl-o-( proportional, variant -hydroxy)tolylphosphate (HO-TOTP), and one ChE inhibitor, 3-benzyl-6-chloro-2-pyrone (3-BCP). The inhibitory effect of CBDP, HO-TOTP, and 3-BCP on FAEE synthase and esterase activity was studied using rat hepatic and pancreatic postnuclear (PN) fractions, commercial porcine hepatic CE and pancreatic ChE, and in HepG2 and rat pancreatic tumor (AR42J) cell lines. Only HO-TOTP and CBDP inhibited FAEE synthase as well as esterase activity of hepatic PN fraction and commercial CE and ChE in a concentration-dependent manner, and the inhibition was found to be irreversible. However, no inhibition was found in pancreatic PN fraction by both TOTP metabolites and 3-BCP. Although 3-BCP inhibited only the esterase activity of commercial ChE in a concentration-dependent manner, the activity was reversible within 30 min of incubation. Studies with HepG2 cells also showed a significant inhibition of FAEE synthase-esterase activity by CBDP and HO-TOTP within 15 min of incubation, while no inhibition was observed in AR42J cells. 3-BCP did not inhibit FAEE synthase-esterase activity either in HepG2 or AR42J cells. Such differential inhibitory effect of the TOTP metabolites on hepatic and pancreatic FAEE synthase-esterase is supported by our earlier in vivo and in vitro studies. Further investigations are needed to understand the biochemical mechanism(s) of inactivation of TOTP metabolites and 3-BCP in the pancreas and AR42J cells towards FAEE synthase-esterase activities.

The contribution of carboxylesterase (CarbE) to toxicity and tolerance to the organophosphorus anticholinesterases (OP-antiChE) paraoxon (diethyl p-nitrophenyl phosphate) and DFP (diisopropylphosphorofluoridate) was investigated in rats. Daily injections (20 days) of paraoxon (0.33 micromol/kg) or DFP (2.72 micromol/kg) reduced AChE activity in brain to 29 or 16% and in diaphragm to 58 or 54%, respectively. The animals tolerated an accumulated 6-fold LD50 dose and survived an LD90 dose of carbachol, indicating tolerance to this cholinergic agonist. A single dose of paraoxon or DFP significantly reduced CarbE activity of plasma, lung and liver. After paraoxon, rapid recovery was seen of plasma and liver CarbE while recovery after DFP was much slower. Daily pretreatment with the CarbE inhibitors CBDP (2-[o-cresyl]-4H-1,2,3-benzodioxa- phosphorin-2-oxide) (7.22 micromol/kg, s.c.) or iso-OMPA (tetraisopropylpyrophosphoramide) (8.76 micromol/kg, i.p.), followed by paraoxon (0.33 micromol/kg, s.c.) 30 min later, prevented the development of tolerance to paraoxon and potentiated its toxicity. Rats died on day four of the combined treatment. The CarbE inhibitors neither potentiated the DFP toxicity, nor prevented tolerance development to DFP. We conclude that rat plasma CarbE provides a significant protection against paraoxon toxicity because its rapid reactivation can reduce the toxicity of repeated paraoxon applications and thus contribute to tolerance development. This same mechanism does not apply to DFP toxicity, as inhibition of CarbE of plasma, liver and lung neither potentiated its toxicity, nor prevented tolerance development. These findings confirm previous observations that CarbE detoxification is of greater importance for highly toxic OP-antiChEs such as nerve agents and paraoxon than for less toxic ones such as DFP.

The contribution of carboxylesterase (CarbE) to the development of tolerance to the organophosphorus anticholinesterase (OP-ANTIChE) paraoxon (diethyl p-nitrophenyl phosphate) was investigated in rats. Daily injections (20 days) of paraoxon (0.09 mg/kg) led to a cumulative dose that was 9.0-fold higher than the acute ED50 of 0.20 mg/kg, s.c. During this period, the rats did not demonstrate visible signs of cholinergic hyperactivity nor did they die, despite the persistence of critically reduced brain acetylcholinesterase (AChE) activity (20-30% of control). In addition, none of these rats died following the administration of a dose of carbachol (3.1 mg/kg, i.p.) that was an LD90 in untreated rats. Daily treatment with the CarbE inhibitors CBDP [2-(o-cresyl)-4H-1,3,2-benzodioxaphosphorin-2-oxide] (2 mg/kg, s.c.) or iso-OMPA (tetraisopropylpyrophosphoramide) (3 mg/kg, i.p.) followed by paraoxon (0.09 mg/kg, s.c.) 60 min later prevented the development of tolerance to paraoxon, since signs of cholinergic hyperactivity were observed and rats died on day 4 of the combined treatment. In tolerant rats, one-time CBDP or iso-OMPA pretreatment increased toxicity to paraoxon, causing the death of all rats within 60 min. The increase in paraoxon toxicity was correlated with inhibition of a plasma CarbE, with high affinity toward alpha-naphthyl acetate (alpha-NA) and to the inhibitors CBDP, iso-OMPA, and paraoxon. Inhibition of a plasma CarbE with high affinity toward p-nitrophenyl acetate (p-NPA) and low affinity to the above inhibitors did not potentiate paraoxon toxicity significantly. Neither the liver CarbEs, which showed high affinity to iso-OMPA, nor the inhibition of butyrylcholinesterase (BCHE) by iso-OMPA in plasma and liver potentiated paraoxon toxicity. By eliminating plasma CarbE (alpha-NA) as potential binding sites for paraoxon with either CBDP or iso-OMPA, paraoxon can exert its toxicity to a greater extent at its specific target site, the functionally important AChE at cholinergic synapses. It is concluded that plasma CarbE (alpha-NA) provided a significant protection against paraoxon intoxication and that the inhibition of this enzyme prevented the tolerance development seen with repeated paraoxon treatments.

OBJECTIVE: To assess the mechanism and rate of in vitro degradation of cisatracurium in aqueous buffer and in human and rat plasma. METHODS: Cisatracurium was incubated in aqueous buffer at various pH values or in human and rat plasma maintained at pH 7.4 with HEPES buffer. Cisatracurium and the degradation products, laudanosine and the monoquaternary alcohol, were quantitated by HPLC with use of fluorescence detection. RESULTS: In Srenson's phosphate buffer, cisatracurium degraded spontaneously by a chemical process commonly referred to as "Hofmann elimination." The rate of degradation increased with increasing pH. From pH 6.4 to 7.8 there was a 6.5-fold increase in the rate of degradation of cisatracurium and, on a molar basis, the final decomposition product laudanosine accounted for all of the drug. At a pH of 7.4, cisatracurium degraded with a half-life of about 34.1 +/- 2.1 minutes. Cisatracurium incubated in human plasma degraded with a mean (+/- SD) half-life of 29.2 +/- 3.8 minutes, which is consistent with Hofmann elimination. Besides laudanosine, and unlike that observed in Srenson's phosphate buffer, significant amounts of the monoquaternary alcohol were formed that slowly degraded to laudanosine. The micromoles of laudanosine formed eventually accounted for the total amount of cisatracurium incubated with human plasma. The monoquaternary alcohol appears to be a product of ester hydrolysis of a monoquaternary acrylate formed during the first step in Hofmann elimination. Evidence for esterase involvement at this step in the degradation of cisatracurium was based on inhibition studies with O-cresyl benzodioxaphosphorin oxide (CBDP), a specific carboxylesterase inhibitor. The addition of CBDP to human plasma completely blocked the formation of monoquaternary alcohol and converted the degradation of cisatracurium to total Hofmann elimination. In rat plasma cisatracurium was hydrolyzed, with a half-life of only 3 1/2 minutes, by carboxylesterases. The addition of CBDP increased the half-life to 25 minutes, which is consistent with Hofmann elimination. CONCLUSION: In human plasma the rate-limiting step in the degradation of cisatracurium is Hofmann elimination, with the initial formation of a monoquaternary acrylate. The observation that the monoquaternary alcohol results from ester hydrolysis of the monoquaternary acrylate by plasma esterase(s) explains the presence of the monoquaternary alcohol metabolite in human plasma during clinical studies with cisatracurium. The rapid hydrolysis of cisatracurium by rat plasma relative to human indicates a major species difference in plasma esterase(s).

2-Substituted-4H-1,3,2-benzodioxaphosphorin 2-oxides (2-substituted-BDPOs) are known to be potent neuropathy target esterase (NTE) inhibitors (I50s for the racemates of 0.2-3 nM) when the 2-substituents are n-alkyl (C5-C12), N-alkoxy (C7-C10), or p-n-alkylbenzyl (C3 and C4). The list of potent inhibitors (I50s < 3 nM) is expanded by the new n-alkylamino (C9) and n-alkylthio (C5, C7, and C9) analogs reported here. The optimal chain length of the 2-substituent is about 10 atoms in the alkylamino and alkylthio series as in our previous study on alkyl and alkoxy moieties. In contrast, an I50 of 60 nM is reported for o-methylphenoxy-BDPO, the neuropathic metabolite of tri-o-cresyl phosphate (TOCP). In addition to substituent effects, each of these compounds contains two enantiomers of unknown stereospecificity as NTE inhibitors. Separation by chiral HPLC with the CHIRALCEL OC column and hexane-2-propanol eluent gives individual enantiomers of > 98% e.e. and a stereospecificity for NTE inhibition depending on the type and chain length of the 2-substituent; e.g., the ratio for inhibitory potency of the individual enantiomers is 1.7-fold for nonylthio, 1255-fold for nonylamino, and 9-fold for the TOCP metabolite. In comparing enantiomeric pairs of BDPOs with alkyl, alkoxy, alkylamino, alkylthio, benzyl, p-butylbenzyl, o-methylphenoxy, or phenyl as the 2-substituent, the more retained enantiomer in HPLC is always the better NTE inhibitor (in a series of twenty-two pairs) and housefly toxicant (based on two pairs) than the less retained one.(ABSTRACT TRUNCATED AT 250 WORDS)

Since pharmacologic treatments of organophosphorus anticholinesterases (OPs) are nearing their practical limit other types of treatment are being sought. One approach is the prophylactic administration of scavengers that will detoxify OPs before they reach their critical target site. We have shown that mice which have been treated with a purified organophosphorus acid anhydride hydrolase (OPAH) are not measurably affected by doses of soman that are lethal to untreated animals. This result indicates that this approach is worthy of exploration and development for protecting military personnel and agriculture workers against OP intoxication.

Saligenin cyclic o-tolyl phosphate (SCOTP) has been proposed as the active metabolite of tri-o-cresyl phosphate (TOCP), a neurotoxic organophosphate. TOCP is also toxic to the testis and SCOTP mimics some of this toxicity. The stability of SCOTP in vivo and its uptake by selected tissues has been measured. Total radioactivity and SCOTP-associated radioactivity were determined in male F-344 rats treated i.v. with 1 mg of [14C]-SCOTP/kg. The half-life of SCOTP in blood was 8.0 +/- 1.1 min. Testes, brain, and muscle had lower concentrations of [14C]-SCOTP-derived radioactivity than blood. Liver and kidney had higher concentrations of radioactivity than blood. HPLC analysis of liver, kidney, testes, and blood extracts showed that 2.8, 48, 11, and 18%, respectively, of the radioactivity present at 5 min was SCOTP. The amount of SCOTP declined rapidly, and at 30 min SCOTP could be detected only in kidney. It appears that SCOTP, although reactive, has sufficient stability to be transported from organ to organ. There is no evidence, however, of active uptake of SCOTP from blood by the testes. Evidence was obtained that SCOTP may act as an alkylating agent.

Pretreatment of rats and guinea pigs with the specific carboxylesterase inhibitor 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide (CBDP) reduces the LD50 of the nerve agent C(+/-)P(+/-)-soman in these species to the same range as in primates. This suggests that such CBDP-pretreated animals can be used in investigations that are relevant for prophylaxis and therapy of intoxication with C(+/-)P(+/-)-soman in primates including humans. In order to test this hypothesis we have studied the toxicokinetics of the toxic C(+/-)P(-)-isomers of soman in artificially respirated and CBDP-pretreated rats and guinea pigs at intravenous doses corresponding to 6x LD50. A comparison of the areas under the curve (AUCs) of the blood levels of C(+/-)P(-)-soman in pretreated and non-pretreated animals at the same absolute dose shows extreme nonlinearity with dose, indicating that CBDP occupies highly reactive binding sites which are no longer available for sequestration of the soman isomers. The AUCs of C(+/-)P(-)-soman at equitoxic doses of 6x LD50 are reduced by pretreatment with CBDP from 1683 to 464 ng.min.ml-1 in rats and from 978 to 176 ng.min.ml-1 in guinea pigs, which is in the range of the AUC in non-pretreated marmosets at an equitoxic dose (419 ng.min.ml-1). The blood levels of the C(+/-)P(-)-isomers in marmosets and CBDP rats are rather similar during the first 7 min, but persist in CBDP rats for 2 h longer at toxicologically relevant levels than in marmosets.(ABSTRACT TRUNCATED AT 250 WORDS)

Diisopropyl phosphorofluoridate (DFP), mipafox, cresylsaligenyl phosphate, and phenylsaligenyl phosphate were applied to a 1.5-cm segment of the common trunk of the sciatic nerve in adult hens. At doses of 18-182 micrograms mipafox and 9-110 micrograms DFP, inhibition of neuropathy target esterase (NTE) for the treated segment was over 80%, whereas for the adjacent distal and proximal segments inhibition was under 40%, 15 min after application. NTE was not affected in the peripheral distal terminations arising from the common sciatic nerve (peroneal branches), contralateral sciatic nerve, brain, and spinal cord. A 24-hr study suggested a displacement of the activity-free region toward more distal segments of the nerve. All animals treated with 55 and 110 micrograms DFP or 110 micrograms mipafox lost a characteristic avian retraction reflex in the treated leg 9-15 days after dosing, suggesting peripheral neurological alterations. Only hens dosed at the maximum dose in both extremities presented alterations in motility (Grade 1 or 2 on a 0-8 scale), suggesting no significant central nervous system alterations. Electron microscopy of peroneal branches showed axon swelling and accumulation of smooth endoplasmic reticulum similar to animals dosed systemically (s.c.) with 1-2 mg/kg DFP. The branches also contained granular and electron-dense materials, as well as some intraaxonal and intramyelinic vacuolization. Clinical effects were not observed in animals protected with a 30 mg/kg (s.c.) dose of phenylmethanesulphonyl fluoride. It is concluded that the peripheral neurological effects of local dosing correlate with the specific modification of NTE in a segment of sciatic nerve and that the axon is a more likely target than the perikaryon or nerve terminal in the triggering mechanism of this axonopathy.

A neurotoxic organophosphate, tri-o-cresyl phosphate (TOCP) is also a testicular toxicant. Histopathologic damage in the testis is first seen in Sertoli cells. TOCP and its activated metabolite saligenin cyclic-o-tolyl phosphate (SCOTP) were evaluated for effects on rat Sertoli cells in primary culture. SCOTP, but not TOCP, caused minor morphologic effects on the cells and increased levels of lactate in the spent medium with no change in pyruvate levels, synthesis of cellular or secreted proteins, or the cyclic AMP response to FSH stimulation. SCOTP was the metabolite of TOCP that produced the largest decrease in nonspecific esterase activity in Sertoli cells (up to 80%), when tested in the concentration range found in vivo. This decrease is consistent with previous in vivo evidence. These in vitro experiments replicate previously observed in vivo biochemical effects and suggest that SCOTP is the metabolite responsible for at least some of the biochemical effects seen in the testis after TOCP exposure.

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., greater than or equal to 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., greater than or equal to 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 (greater than or equal to 4 microns) 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.

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.

Rodents are relatively insensitive to the neurotoxic effects of various organophosphorus compounds. The purpose of this investigation was to determine if differences in inactivation of CBDP could explain the strain differences in the sensitivity to neurotoxicity following administration of TOCP (tri-o-cresyl phosphate) observed by Carrington and Abou-Donia (1988). Serum carboxylesterase but not cholinesterase is an important detoxification route for organophosphates. Serum carboxylesterase and cholinesterase activity were significantly different (p less than 0.05) among the various strains of rats. The rank order of carboxylesterase activity was Sprague Dawley (6158 nmole/ml serum/min) greater than Long Evans (5589) greater than Fischer 344 (5010) whereas the rank order for cholinesterase activity was Fischer 344 greater than Sprague Dawley greater than Long Evans. TOCP is metabolized to the active neurotoxicant CBDP (2-/o-cresyl/4H:1:3:2-benzodioxaphosphorin-2-oxide). The ED50 for CBDP inhibition of serum carboxylesterase activity was found to vary considerably for the various strains of rats. The rank order of CBDP ED50 concentration in the various strains was Fischer 344 (437 microM) greater than Long Evans (339 microM) greater than Sprague Dawley (78 microM), indicating that there was a difference between the carboxylesterase of the various strains with regard to interaction with CBDP. It is suggested that the differences in the quantity of serum carboxylesterase combined with the differences in the interaction of the inhibitor with the enzyme(s) may be responsible for the strain differences observed by Carrington and Abou-Donia (1988).

The toxicity of the organophosphorus poison soman (pinacolylmethylphosphonofluoridate) is attributable to its irreversible inhibition of the enzyme acetylcholinesterase. In addition, soman binds irreversibly to a number of noncholinesterase tissue binding sites which appear to be its major means of in vivo detoxification. This study was conducted to determine the hepatic subcellular localization of these sites. Subcellular fractions of liver from male Sprague-Dawley rats (200-250 g) were prepared by differential and isopycnic density gradient centrifugation. The binding of [14C]soman to these subcellular fractions was determined in the presence and absence of cresylbenzodioxaphosphorin oxide (CBDP), a compound that binds irreversibly to the noncholinesterase soman binding sites. Crude fractionation of liver homogenates into nuclear, mitochondrial, microsomal, and soluble fractions revealed that 78% of the total CBDP-sensitive binding activity was localized in the nuclear and microsomal fractions. Further purification of these fractions indicated that all of the homogenate binding activity could be accounted for in the purified microsomal fraction. When purified liver microsomes were solubilized and fractionated on linear sucrose gradients, 90% of the CBDP-sensitive soman binding activity cosedimented with carboxylesterase activity which suggests that these binding sites are carboxylesterase.

Soman poisoning presents a problem in terms of its detailed pathophysiology and its detoxification mechanism(s). The present study was designed to evaluate the role of carboxylesterases (CaE) and cholinesterase (ChE) in the distribution and detoxification of soman in vivo. Mice were injected (i.v.) with 0.06-1.0 LD50 of [3H]-soman, 60 min following pretreatment with either 2-O-cresyl-4H-1:2:3 benzodioxa-phosphorine-2-oxide (CBDP), which blocks CaE or 7-(methylethoxyphosphinyloxy)-1-methyl quinolinium iodide (MEPQ), which selectively inhibits intravascular ChE. One hour after [3']-soman administration animals were sacrificed and whole body autoradiography was performed. High concentrations of [3H]-soman were found in lung and kidney in control mice, and low concentrations were found in central nervous system. Pretreatment with CBDP caused a 93% decrease in radioactive labelling in the lung, and a minor decrease in overall labelling, whereas pretreatment with MEPQ did not change the distribution pattern of [3H]-soman. It is concluded that lung is a major target organ for soman detoxification and that it exerts this effect by means of enzymatic reaction with soman through the abundant amounts of CaE which are present in the lung. Intravascular ChE has little (if any) effect on the distribution and detoxification of soman in vivo.

The effect of the microsomal enzyme inducer beta-naphthoflavone (beta NF) on the development of organophosphorus-induced delayed neuropathy (OPIDN) was examined in two laboratories (VPI and MSU), utilizing two strains of White Leghorn hens. A single intraperitoneal injection of beta NF at 80 mg/kg body weight 48 h prior to administration of o-tolyl saligenin phosphate (TSP), the neuroactive metabolite of tri-o-tolyl phosphate (TOTP), caused a significant increase in hepatic microsomal cytochrome P-450 concentrations and aniline hydroxylase activities after 72 h in both strains. Hepatic carboxylesterase and cholinesterase activities were not affected by beta NF treatment in either strain. Administration of TSP in single subcutaneous doses of 20 and 25 mg/kg body weight (VPI) or 30 and 60 mg/kg body weight (MSU) caused significant inhibition of whole-brain neuropathy target esterase (NTE) activity 24 h postdosing, and hens subsequently developed clinical signs characteristics of OPIDN. beta NF had no significant effect on NTE inhibition or on initiation or severity of OPIDN clinical signs. However, OPIDN clinical signs were less severe in the strain of bird (MSU) with the higher intrinsic hepatic carboxylesterase activity and the higher beta NF-induced cytochrome P-450 concentration. The study indicates that microsomal enzyme induction, which has been shown to alleviate TOTP-induced delayed neuropathy, could not alleviate OPIDN resulting from exposure to TSP. This study also suggests that strain may affect susceptibility to TSP-induced delayed neuropathy.

The dose-response (0.1 to 1000 mg/kg sc) effects of 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide (CBDP; a metabolite of the organophosphorus compound tri-o-cresylphosphate) on total cholinesterase (ChE) and carboxylesterase (CaE) activities in tissues from the rat were examined. Doses of CBDP greater than 1.0 mg/kg inhibited CaE activity maximally (greater than 99%) in plasma and lung, two important sites for detoxification of organophosphorus toxicants. A biphasic dose-dependent inhibition of ChE activity was seen in all tissues; the ED50 values showed a difference of two orders of magnitude between the first and the second phases of the dose-response curves. CBDP inhibited the blood esterases in the order plasma CaE much greater than plasma ChE much greater than red blood cell (RBC) ChE. The biphasic dose-response curve and preferential inhibition of the blood esterases may reflect the inhibition of butyrylcholinesterase in preference to acetylcholinesterase in these tissues. At doses of CBDP below 1.0 mg/kg, plasma, RBC, and brain regional ChE activities were inhibited by less than 10%, whereas at doses above 2.0 mg/kg, ChE activities were inhibited substantially (up to 80% in plasma, up to 60% in RBC, and greater than 90% in brain regions). On the basis of these results, a dose of CBDP between 1.0 and 2.0 mg/kg should prove useful as a pretreatment for studies of OP toxicity in the rat.

The toxicity of soman was investigated in the rat with and without pretreatment with cresylbenzodioxaphosphorin oxide (CBDP). Without pretreatment, the 24-h LD50 for soman was 118.2 micrograms/kg s.c., and soman inhibited carboxylesterase (CaE) activity in plasma (ED50 of 55 micrograms/kg) and cholinesterase (ChE) activity in brain regions (ED50 values of 65-105 micrograms/kg) in a dose-related manner. With pretreatment, the 24-h LD50 for soman was reduced by approximately 6-fold and 8-fold (by 1.0 mg/kg and 16.0 mg/kg of CBDP, respectively), and the ED50 values for soman-induced inhibition of ChE activity in brain regions were reduced by approximately 10-fold (by 1.0 mg/kg of CBDP). The dose-dependent severity of soman intoxication varied widely in rats treated with soman alone but not in CBDP-pretreated rats, and the ED50 for the occurrence of signs of soman intoxication was reduced approximately 7-fold following CBDP (1.0 mg/kg) pretreatment. These data support the hypothesis that CBDP pretreatment effectively blocks tissue CaE sites which serve to detoxify soman, thus potentiating both the soman-induced inhibition of ChE in the CNS and the lethality of soman.

The ability of the carbamates pyridostigmine and physostigmine to protect against the lethal effects of soman, an extremely toxic anticholinesterase agent, was measured in rats, guinea pigs and rabbits. Pharmacologically equivalent doses of these carbamates that inhibited 70% of the blood acetylcholinesterase in each species were injected i.m. 25 min before s.c. injection with soman. Pretreatment with either carbamate, in combination with 17.4 mg/kg of atropine, produced protection against soman toxicity in all species. When protection was expressed as the ratio between the soman LD50 values in carbamate-protected animals and control animals, this protective ratio varied 3-fold between species (2.1-6.1 for pyridostigmine; 2.2-6.6 for physostigmine). When protection was expressed as the difference in the soman LD50 values between carbamate-protected animals and control animals, this protective difference was consistent among species (126 +/- 19 micrograms/kg). Species variation in protective ratios was observed largely because the control LD50 values defining soman toxicity in unprotected animals varied among species (20 micrograms/kg in rabbits, 28 micrograms/kg in guinea pigs and 126 micrograms/kg in rats). The species variation of the soman LD50 values in control animals was eliminated by pretreating animals with cresylbenzodioxaphosphorin oxide, which reduced the species variation in soman detoxification. The LD50 values for soman in cresylbenzodioxaphosphorin oxide-treated animals (9.8-15.6 micrograms/kg) did not differ significantly between species. Similarly, protective ratios for carbamates against soman in cresylbenzodioxaphosphorin oxide-treated animals were also clustered in a narrow range (8.5-11.4 for pyridostigmine; 9.0-13.4 for physostigmine) that did not differ significantly, regardless of species or carbamate.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The interaction of C(+/-)P(+/-)-soman (pinacolyl methylphosphonofluoridate) and its individual stereoisomers with serum carboxylic-ester hydrolase and potentiation of their toxicity by a carboxylic-ester hydrolase inhibitor CBDP (2-(2-methylphenoxy)-4H-1,3,2-benzodioxaphosphorin-2-oxide) was investigated. C(+/-)P(+/-)-Soman and the individual stereoisomers all inhibited purified mouse serum carboxylic-ester hydrolase to different degrees. C(+/-)P(+/-)-Soman and the C(-)P(-)- and C(+)P(-)-isomers had Ki values of 30.6, 18.7, and 35.7 nM, respectively, and C(-)P(+)- and C(+)P(+)-isomers had Ki values of 1412 and 2523 nM, respectively. In toxicity experiments CBDP (0.5 mg/kg; iv 1 hr prior to soman) pretreatment potentiated the toxicity of C(+/-)P(+/-)-, C(+)P(-)-, and C(-)P(-)-soman to a similar degree. Thus, it appears that the toxic stereoisomers of soman have a similar affinity for mouse serum carboxylic-ester hydrolase, and CBDP pretreatment does not enhance selectively the toxicity of one stereoisomer over the other.

Subcutaneous administration of 2 mg/kg cresylbenzodioxaphosphorin oxide (CBDP) produced complete inhibition of carboxylesterase activity in plasma and lung of mice, rats, guinea pigs and rabbits, without inhibition of acetylcholinesterase activity in either brain or diaphragm. This CBDP treatment also reduced the subcutaneous soman LD50 in these species by 48-90% in comparison to the soman LD50 in control animals. The interspecies differences in the soman LD50 values that were seen in control animals were absent in CBDP-treated animals. The soman LD50 values in control animals were 125 micrograms/kg (mouse), 116 micrograms/kg (rat), 32.3 micrograms/kg (guinea pig) and 22.8 micrograms/kg (rabbit), whereas the soman LD50 values in CBDP-treated animals from these species were clustered in a narrow dose range (11.8-15.6 micrograms/kg) and were not significantly different. This suggests that the amount of CBDP-sensitive carboxylesterase available for detoxification of soman in each species may be an important determinant of interspecies differences in soman toxicity.

Various doses of CBDP (2-(2- methylphenoxy )-4H-1,3,2- benzodioxaphosphorin -2-oxide), a metabolite of tri-o-cresyl phosphate, increased dramatically the acute toxicity of soman ( pinacolyl methylphosphonofluoridate ) in mice. CBDP (5 mg/kg; iv) reduced the soman LD50 value from 136 micrograms/kg in control to 6.95 micrograms/kg. The potentiation of soman toxicity following CBDP pretreatment appeared to be due primarily to inhibition of plasma aliesterase activity. Inhibition of liver aliesterase was not of primary importance in the potentiation of soman toxicity following CBDP pretreatment. In addition pretreatment with ISO-OMPA ( tetraisopropyl pyrophosphoramide ), a selective inhibitor of pseudocholinesterase, had no effect on the acute toxicity of soman. Similarly pretreatment of mice with pyridostigmine, a quaternary carbamate anticholinesterase which does not inhibit aliesterase , resulted in marked inhibition of diaphragm, plasma, and brain acetylcholinesterase had no effect on the acute toxicity of soman. Plasma aliesterase may be a depot for soman poisoning. The acute toxicity of soman by the ip, sc, and iv routes of administration was reduced following pretreatment of mice with phenobarbital (100 mg/kg) for 4 days. The reduced toxicity of soman following phenobarbital pretreatment was due to induction of liver aliesterase activity which subsequently resulted in an increase in plasma aliesterase activity. Thus more soman was probably bound to plasma aliesterase activity resulting in a reduction in acute toxicity of soman. Conversely pretreatment of mice with pentobarbital (70 mg/kg; ip) increased the toxicity of soman. This was probably the result of inhibition of plasma aliesterase by pentobarbital pretreatment combined with the central respiratory depression following pentobarbital administration. Following pentobarbital pretreatment soman inhibition of brain acetylcholinesterase was increased suggesting that plasma aliesterase inhibition alters the distribution of free soman in vivo. In summary, in mice plasma aliesterase appears to be an extremely important detoxification route for soman in vivo.

CBDP (2-/O-cresyl/4H:1:2-benzodioxaphosphorin-2-oxide) pretreatment produced a dramatic increase in the toxicity of soman in mice following the subcutaneous (s.c.) or intraperitoneal (i.p.) route of administration. This increase in soman toxicity was very highly correlated with inhibition of plasma aliesterase activity. Other enzymes (e.g. liver aliesterase and plasma cholinesterase) were inhibited by CBDP pretreatment; however, they did not appear to play a significant role in the potentiation of soman toxicity by CBDP. Liver aliesterase was not inhibited by doses of CBDP which produced significant increases in soman toxicity. Similarly, doses of Iso-OMPA, a selective inhibitor of pseudocholinesterase, which completely inhibited plasma cholinesterase, had no effect on soman toxicity. Pyridostigmine pretreatment which inhibited brain, diaphragm and plasma acetylcholinesterase 27, 57 and 60%, respectively, while not inhibiting plasma aliesterase, did not affect soman toxicity. The results of this study demonstrate that, in mice, plasma aliesterase is an extremely important detoxification route for soman.

Pretreatment of mice with atropine (17.4 mg/kg) + NaF (5 or 15 mg/kg) had a significant antidotal effect over atropine alone against the lethality produced by soman and sarin. Atropine + NaF (15 mg/kg) was effective against tabun, whereas the lower dose of NaF was not. An effect of NaF on organophosphate inhibited acetylcholinesterase could not account for the antidotal action of NaF. NaF had no effect on liver somanase activity but inhibited aliesterase activity. Aliesterase activity in NaF pretreated soman-poisoned mice was significantly (p less than 0.05) higher than in those receiving atropine alone. In CBDP-pretreated mice NaF did not significantly attenuate the toxicity of soman. It is hypothesized that the antidotal effect of NaF versus organophosphate poisoning is due to its antidesensitizing action at nicotinic receptors in the neuromuscular junction and/or sympathetic ganglia in addition to the proposed increased hydrolysis of sarin and direct detoxification of tabun.

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.

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"