Tri-o-cresyl Phosphate Administration Research Articles

Verapamil abolished the enhancement of protein phosphorylation of brainstem mitochondria and synaptosomes from the hens dosed with tri- o-cresyl phosphate

To explore the changes of the endogenous phosphorylation of brainstem mitochondrial and synaptosomal proteins in adult hens dosed with tri- o-cresyl phosphate (TOCP) following the development of organophosphate-induced delayed neurotoxicity (OPIDN). Verapamil (7 mg/(kg day), i.m.) was given for 4 days. A dose of TOCP (750 mg/kg, p.o.) was administrated in second day after verapamil. Phosphorylation of the proteins from brainstem mitochondria and synaptosomes was assayed in vitro by using [γ- 32P]ATP as phosphate donor. Radiolabeled proteins were separated by SDS-PAGE and visualized by autoradiography. The results showed that TOCP administration enhanced the phosphorylation of the cell organelle proteins (mitochondria: 60, 55, 45, and 20 kDa; synaptosomes: 65, 60, and 20 kDa), while verapamil abolished the enhancement induced by TOCP. Additionally, the reaction for the phosphorylation is catalyzed by the calcium/calmodulin protein kinase. Therefore, TOCP can enhance the phosphorylation of the brainstem mitochondrial and synaptosomal proteins from the hens with OPIDN; however, protection from the enhancement of the phosphorylation should be involved in the mechanisms of the amelioration of TOCP-induced delayed neurotoxicity by verapamil.

Effect of tri- o-cresyl phosphate and methamidophos on 45Ca uptake by brain synaptosomes in hens

The effect of tri- o-cresyl phosphate (TOCP) and methamidophos (MET) on potassium-stimulated 45Ca uptake by brain synaptosomes in hens was studied. An in vivo test showed that TOCP increased potassium-stimulated calcium uptake 2 h after its administration, but that verapamil suppressed the enhancement of this calcium uptake. An in vitro test showed that lower concentrations of TOCP stimulated calcium uptake by synaptosomes, but that higher concentrations inhibited the uptake. In contrast, all tested concentrations of MET obviously inhibited calcium uptake; however, since calcium uptake was decreased by the administration of verapamil plus either TOCP or MET, the mechanism by which TOCP affects the voltage-operated calcium channel may be different from that of MET. The disruption of calcium homeostasis may be involved in organophosphate-induced delayed neurotoxicity (OPIDN). Calcium channel blocker may ameliorate OPIDN by maintaining calcium homeostasis in nerve cells.

Effect of Verapamil on Changes in Brain Protein Phosphorylation after Oral Administration of Tri-O-cresyl Phosphate to Hens

The endogenous phosphorylation of specific brain proteins in white Leghorn laying hens treated with tri-o-cresyl phosphate (TOCP) with and without verapamil, a calcium channel blocker, which has been shown to ameliorate clinical signs and nervous lesions associated with organophosphate-induced delayed neuropathy (OPIDN), was determined. Verapamil was given im at the dose of 7 mg/kg/day for 4 days beginning 1 day before administration of TOCP (750 mg/kg, po). Phosphorylation of crude synaptosomal (P2) membrane proteins was assayedin vitrousing [γ-32P]ATP as a phosphate donor. Following resolution of brain membrane proteins by SDS-PAGE, specific protein phosphorylation was detected by autoradiography and quantified by densitometry TOCP caused, 21 days after dosing, an enhancement of the phosphorylation of membrane proteins with apparent molecular weights of 40, 55, and 60 kDa by as much as 253, 322, and 609%, respectively. Verapamil administered to TOCP-treated hens abolished the enhancement and even caused some inhibition. This indicates that calcium channel blocker may contribute to the amelioration of OPIDN by preventing the enhancement of phosphorylation of brain membrane proteins.

Serum carboxylesterase activity in various strains of rats: sensitivity to inhibition by CBDP (2-/o-cresyl/4H∶1∶3∶2-benzodioxaphosphorin-2-oxide)

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 interaction of Sertoli and Leydig cells in the testicular toxicity of tri- o-cresyl phosphate

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.

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

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

Reproductive tract lesions resulting from subchronic administration (63 days) of tri- o-cresyl phosphate in male rats

An initial dose-range pilot study where animals were gavaged with between 100 and 1600 mg tri- o-cresyl phosphate (TOCP)/kg/day for 14 days resulted in decreased epididymal sperm density and disruption of the seminiferous epithelium in 100% of treated animals. A subchronic 63-day study (reflecting the 49-day length of the rat seminiferous epithelium cycle plus the 14-day transit time of spermatids through the epididymis was initiated. Dose-dependent (10 to 100 mg TOCP/kg/day) decreases in cauda epididymal sperm motility and density, testicular enzyme activities, and alterations in sperm morphology were observed. Concurrent pair-fed controls (matched to the highest dose group, 100 mg TOCP/kg/day) indicated that weight loss resulting from TOCP administration had minimal contributory effects to the testicular toxicity seen. Plasma α-tocopherol acetate (vitamin E) and testosterone concentrations were unaffected. Tri- p-cresyl phosphate (TPCP), the nonneurotoxic structural analog of TOCP, produced no toxic effects, demonstrating the necessity of the ortho-cresol moiety for induction of damage. A minimum effective (threshold) dose for observable testicular toxicity was determined to be 10–25 mg TOCP/kg in this study. These data suggest that TOCP interferes with spermatogenic processes and sperm motility directly and not via an androgenic mechanism or decreased vitamin E availability.

Changes in in vitro brain and spinal cord protein phosphorylation after a single oral administration of tri-o-cresyl phosphate to hens.

The effect of a single oral 750 mg/kg dose of tri-o-cresyl phosphate (TOCP) on the endogenous phosphorylation of brain and spinal cord proteins was assessed in hens during the development of and recovery from delayed neurotoxicity. Crude membrane and cytosolic fractions were prepared from the brains and spinal cords of control and TOCP-treated hens at 1, 7, 14, 21, 35, and 55 days after treatment. Brain and spinal cord protein phosphorylation with [gamma-32P]ATP was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), autoradiography, and microdensitometry. TOCP administration conferred calcium and calmodulin dependence on the phosphorylation of a few brain cytosolic proteins and caused an increase in the phosphorylation of a number of other cytosolic and membrane proteins. This effect of TOCP was large in magnitude, and its time course reflected the onset of and recovery from the signs of ataxia and paralysis associated with delayed neurotoxicity in the hen. The molecular weights (Mr) and maximal phosphorylation (percent of control) for the most prominently affected bands were as follows: brain cytosol--50K (183%), 55K (575%), 60K (529%), 65K (273%), and 70K (548%); brain membranes--50K (622%) and 60K (697%); and spinal cord cytosol--20K (182%). The role of endogenous phosphorylation reactions in and their potential usefulness as biochemical indicators of delayed neurotoxicity are being explored further.

Effect of oral administration of tri-o-cresyl phosphate on in vitro phosphorylation of membrane and cytosolic proteins from chicken brain.

The effects of a single oral dose of 750 mg/kg tri-o-cresyl phosphate (TOCP) on the endogenous phosphorylation of specific brain proteins were assessed in male adult chickens following the development of delayed neurotoxicity. Phosphorylation of crude synaptosomal (P2) membrane and synaptosomal cytosolic proteins was assayed in vitro by using [gamma-32P]ATP as phosphate donor. Following resolution of brain proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis, specific protein phosphorylation was detected by autoradiography and quantified by microdensitometry. TOCP administration enhanced the phosphorylation of both cytosolic (Mr 65,000 and 55,000) and membrane (20,000) proteins by as much as 146% and 200%, respectively.

Effect of tri-O-cresyl phosphate (TOCP) poisoning on sensory nerve terminations of slow loris (Nycticebus coucang coucang).

Sensory nerve terminations in the oral and digital tissues of slow loris (Nycticebus coucang coucang) were studied after topical or systemic administration of TOCP. Histochemical studies revealed a reduction of AChE and complete absence of ChE in sensory nerve terminations. In electron microscopy the light cells of taste buds showed vacuolization, degenerating mitochondria, distended endoplasmic reticulum, vesicle formation and “cytosegregosomes” while dark cells also has unusual shaped dense core granular vesicles (1,200–2,500 A diameter). The axons were “ballooned” with fragmenting mitochondria, some agranular vesicles and disorientated neurotubules lying in a dispersed, lightly granular axoplasm. Moreover, the Nauta-Gygax method (1954) revealed degenerative changes in the digital sensory end-organs as well as in the distal portion of the sensory nerve fibres. The significance of these findings is discussed.

Lipid changes in tri- o-cresylphosphate-induced neuropathy

The possible involvement of lipids in the production of the delayed neuropathy induced by tri- o-cresylphosphate (TOCP) has not been resolved although this condition has been extensively studied because of its possible relationship to multiple sclerosis. We have found that two changes occur in hens just before and at the time paralysis develops. (1) The major sciatic nerve phospholipids become more readily hydrolyzable by the phospholipases of Agkistrodon p. piscivorus venom (cottonmouth moccasin). This phenomenon was not observed in hen brain, where lesions have not been reported, nor in the sciatic nerve of rats, a nonsusceptible species. (2) There is a rise of 25–50% in the cholesterol level of hen sciatic nerve and a 50% decrease in its triglyceride content. All the other parameters studied (distribution of phospholipids, diglycerides, cholesterol esters, proteolipids, and tissue phospholipases) are not affected by TOCP treatment. We have also followed in hens the metabolism of TOCP and the fate of its neurotoxic metabolites. Liver is very active in converting the organophosphate to the anticholinesterase metabolites, whereas brain is only 3% as active, and spinal cord and sciatic nerve are inactive. This in vitro conversion is inhibited completely by 5 × 10 −4 m 2-diethylaminoethyl-2,2-diphenylvalerate (SKF 525-A); however, SKF 525-A did not prevent the onset of paralysis in vivo. We did not detect the in vivo presence of the liposoluble anticholinesterase metabolites of TOCP in the nervous tissues studied, although frequent measurements were made for 5 days after administration of TOCP. A possible explanation for tissue and species sensitivity to organophosphate-induced neuropathy is proposed.

Enzymatic and antidotal studies on the neurotoxic effect of certain organophosphates

The antiesterase activity of tri- o-cresyl phosphate (TOCP) and its cyclic phosphate metabolite (SM-1) was examined. Administration of TOCP and SM-1 to hens in ataxia-inducing doses resulted in selective rather than prolonged inhibition of the nerve esterases hydrolyzing butyryl esters of choline, glycerol, and phenols. Nerve esterase inhibition was more persistent with TOCP than with SM-1. Selective inhibition of esterases hydrolyzing butyryl esters was also evident in studies in vitro with spinal cord homogenates and SM-1 or di isopropyl fluorophosphate (DFP). TOCP and SM-1 did not affect succinate, α-ketoglutarate, or pyruvate oxidation by preparations of nerve and liver from treated birds, nor was the response of the pyruvate oxidation by brain homogenates to thiamine pyrophosphate affected by TOCP. Many compounds tested as possible antidotes for the neurotoxicity induced by TOCP, SM-1, and DFP proved ineffective. Cortisone acetate gave limited improvement in the condition of the birds, thiamine and oxythiamine appeared to be detrimental, and many potential cholinesterase reactivators and other materials were without effect on the neurotoxicity.

Immune reactions induced in infants by intestinal absorption of incompletely digested cow's milk protein

The possible involvement of lipids in the production of the delayed neuropathy induced by tri-o-cresylphosphate (TOCP) has not been resolved although this condition has been extensively studied because of its possible relationship to multiple sclerosis. We have found that two changes occur in hens just before and at the time paralysis develops. (1) The major sciatic nerve phospholipids become more readily hydrolyzable by the phospholipases of Agkistrodon p. piscivorus venom (cottonmouth moccasin). This phenomenon was not observed in hen brain, where lesions have not been reported, nor in the sciatic nerve of rats, a nonsusceptible species. (2) There is a rise of 25–50% in the cholesterol level of hen sciatic nerve and a 50% decrease in its triglyceride content. All the other parameters studied (distribution of phospholipids, diglycerides, cholesterol esters, proteolipids, and tissue phospholipases) are not affected by TOCP treatment.We have also followed in hens the metabolism of TOCP and the fate of its neurotoxic metabolites. Liver is very active in converting the organophosphate to the anticholinesterase metabolites, whereas brain is only 3% as active, and spinal cord and sciatic nerve are inactive. This in vitro conversion is inhibited completely by 5 × 10−4 m 2-diethylaminoethyl-2,2-diphenylvalerate (SKF 525-A); however, SKF 525-A did not prevent the onset of paralysis in vivo. We did not detect the in vivo presence of the liposoluble anticholinesterase metabolites of TOCP in the nervous tissues studied, although frequent measurements were made for 5 days after administration of TOCP. A possible explanation for tissue and species sensitivity to organophosphate-induced neuropathy is proposed.