Effects Of Paraoxon Research Articles

Effects of organophosphates on cholinesterase activity and neurite regeneration in Aplysia

In Aplysia, a marine mollusc, acetylcholinesterase (AChE) is present in cholinergic and non-cholinergic neurons and in hemolymph. Aplysia hemolymph has a very high level of AChE which promotes neurite growth in primary cultures of dopaminergic neurons via a non-catalytic mechanism. In contrast, AChE is known to facilitate neurite growth in cholinoceptive neurons by hydrolyzing ACh which inhibits neurite growth. In order to test whether AChE’s site-specific neurotrophic action varies with the neuronal phenotype, we investigated the effects of active-site inhibited hemolymph AChE on neurite growth of cholinergic neurons of Aplysia in primary culture. Organophosphates being long-acting active site inhibitors of AChE were chosen for this study. The effects of active site inhibited hemolymph AChE was tested on large cholinergic neurons, R2 (abdominal ganglion) and LPL1 (left pleural ganglion) as well as small cholinergic neurons (buccal ganglion) of Aplysia, maintained in culture. Partially purified hemolymph AChE was inhibited by either 10 μM of echothiophate or 5 μM of paraoxon. Neurons were maintained in (1) L15 (defined medium) alone; (2) L15+echothiophate; (3) L-15+paraoxon; (4) L-15+hemolymph AChE; (5) L15+hemolymph AChE+echothiophate; and (6) L-15+hemolymph AChE+paraoxon. Addition of uninhibited hemolymph AChE significantly increased neurite growth of cultured neurons compared to L15 alone. In the presence of echothiophate-inhibited or praoxon-inhibited AChE, neurite growth was significantly reduced when compared to L15+uninhibited AChE. While the presence of echothiophate by itself did not reduce survival or neurite growth when compared to L-15 alone, the presence of paraoxon by itself markedly reduced survival and neurite growth of cultured neurons. The results show that AChE’s catalytic action contributes to enhance neurite growth in cholinergic neurons and the effects of paraoxon appears to differ from that of echothiophate on cholinergic neurons of Aplysia.

Acyloxyalkoxy-based cyclic prodrugs of opioid peptides: evaluation of the chemical and enzymatic stability as well as their transport properties across Caco-2 cell monolayers.

To evaluate the chemical and enzymatic stability, as well as the cellular permeation characteristics, of the acyloxyalkoxy-based cyclic prodrugs 1 and 2 of the opioid peptides [Leu5]-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively. The rates of conversion of 1 and 2 to [Leu5]-enkephalin and DADLE, respectively, were measured by HPLC in HBSS, pH = 7.4, and in various biological media (e.g., human plasma and Caco-2 cell and rat liver homogenates) having measurable esterase activity. The cellular permeation and metabolism characteristics of [Leu5]-enkephalin, DADLE and the cyclic prodrugs 1 and 2 were measured using Caco-2 cell monolayers grown onto microporous membranes and monitored by HPLC. Cyclic prodrugs 1 and 2 degraded slowly but stoichiometrically to [Leu5]-enkephalin and DADLE, respectively, in HBSS, pH = 7.4. In homogenates of Caco-2 cells and rat liver, as well as 90% human plasma, the rates of disappearance of the cyclic prodrugs were significantly faster than in HBSS. The stabilities of the cyclic prodrugs 1 and 2 were increased significantly in 90% human plasma and Caco-2 cell homogenates when paraoxon, a potent inhibitor of serine-dependent esterases, was included in the incubation mixtures. A similar stabilizing effect of paraoxon was not observed in 50% rat liver homogenates, but was observed in 10% homogenates of rat liver. When applied to the AP side of a Caco-2 cell monolayer, DADLE and cyclic prodrugs 1 and 2 exhibited significantly greater stability than [Leu5]-enkephalin. Based on their physicochemical properties (i.e., lipophilicity), cyclic prodrugs 1 and 2 should have exhibited high permeation across Caco-2 cell monolayers. Surprisingly, the AP-to-BL apparent permeability coefficients (P(App)) for cyclic prodrugs 1 and 2 across Caco-2 cell monolayers were significantly lower than the P(App) value determined for the metabolically stable opioid peptide DADLE. When the P(App) values for cyclic prodrugs 1 and 2 crossing Caco-2 cell monolayers in the BL-to-AP direction were determined, they were shown to be 36 and 52 times greater, respectively, than the AP-to-BL values. Cyclic prodrugs 1 and 2, prepared with an acyloxyalkoxy promoiety, were shown to degrade in biological media (e.g., 90% human plasma) via an esterase-catalyzed pathway. The degradation of cyclic prodrug 1, which contained an ester formed with an L-amino acid, degraded more rapidly in esterase-containing media than did prodrug 2, which contained an ester formed with a D-amino acid. Cyclic prodrugs 1 and 2 showed very low AP-to-BL Caco-2 cell permeability, which did not correlate with their lipophilicities. These low AP-to-BL permeabilities result because of their substrate activity for apically polarized efflux systems.

Acetylcholine prevents toxic effects of paraoxon on mouse sperm.

Acetylcholine prevents toxic effects of paraoxon on mouse sperm.

Antagonism of the Lethal Effects of Paraoxon by Carrier Erythrocytes Containing Phosphotriesterase

Antagonism of the Lethal Effects of Paraoxon by Carrier Erythrocytes Containing Phosphotriesterase

Effect of paraoxon and sulfhydryl reagents on3H-quinuclidinol benzoate and3H-methylscopolamine with synaptic membranes of rat brain

Paraoxon is shown to reduce the density of M-choline receptors in the cortex of rat brain. Inhibition of3H-QNB is noncompetitive and reversible in nature. Sulfhydryl reagents do not affect this process. The mechanism of action of PO is thought to include direct effects on brain muscarine receptors.

Anticholinesterase activity measurement by a choline biosensor: application in water analysis

A choline amperometric biosensor was assembled and used to measure the anticholinesterase activity due to compounds (which have the property to inhibit cholinesterase enzymes) present in water samples. This parameter can be used as a ‘toxicological index’, defined as the amount of compound which causes a certain percentage of cholinesterase inhibition equivalent to a known amount of a reference compound causing the same percentage inhibition. The organophosphorus insecticide Paraoxon, which has proved to be a strong inhibitor of cholinesterase enzymes, was chosen as the reference compound. The analysis was carried out by monitoring the decrease of cholinesterase activity in the presence of a pesticide and a substrate specific for the enzyme whose reaction produces choline. The decrease in choline production was measured by the choline sensor and correlated to the concentration of anticholinesterase compound present in the solution. Parameters such as buffer, pH, temperature and incubation time were optimized. The rate constant K i was calculated experimentally for Paraoxon and used in the anticholinesterase activity measurements at different fixed incubation times. The probe was calibrated with different standard solutions of Paraoxon. The effect of Paraoxon and heavy metals on the choline probe was evaluated. This probe was then used for the determination of anticholinesterase activity of some organophosphorus pesticides, and heavy metals in spiked waters. Samples were also analysed by liquid/liquid extraction and GC determination. Results seem to correlate with acute toxicity expressed as LD 50 (oral, rat). Analysis of water samples from different sources in central Italy were analysed for total anticholinesterase activity (TAA) and compared with a reference procedure.

Toxic response to paraoxon is accompanied by an increased rate of acute-phase protein synthesis

The effect of paraoxon (1 and 2 LD 50) on the rate of acute phase protein (APP) synthesis has been examined. In the liver of paraoxon-administered rats transcription of the APP genes and synthesis of the corresponding mRNAs and proteins proceeded at a remarkably higher rates than in the controls. The plasma APP level increased with time, reaching a maximum 24 h after treatment. At this time point the plasma concentration of α 1-acid glycoprotein was eightfold and that of hemopexin negligibly above the control value, whereas the magnitude of increase in the levels of cysteine protease inhibitor, fibrinogen, α 2-macroglobulin, and haptoglobin ranged from six- to twofold. Alike to inflammatory agents, the paraoxon induced an early elevation in the plasma corticosterone level and the immunosuppressive activity of the serum which persisted after the hormone level has returned to control value. Analogy between the response to intoxication with organophosphates and the acute phase reaction to tissue injury is discussed.

Effects of paraoxon on spike initiation and conduction block in the giant interneurons of the American cockroach, Periplaneta americana

1. The effects of paraoxon were studied on spike initiation and conduction in the giant interneurons (GIs) of the American cockroach, using electrophysiological techniques. 2. Paraoxon treatment induced high-frequency bursts in GI axons. During these bursts, overshooting spikes recorded in the sixth abdominal ganglion were replaced, in phase, by small, decrementai potentials. 3. These small potentials were not EPSPs since current injection could modulate their frequency. 4. An analysis of anteriorly conducted spikes indicates that the site of spike initiation is located near the dendritic region of the GI and is unchanged by paraoxon treatment.

Effects of paraoxon, p-nitrophenol, phenyl saligenin cyclic phosphate, and phenol on the rat interleukin 2 system

Two organophosphorus compounds, paraoxon and phenyl saligenin cyclic phosphate, as well as p-nitrophenol and phenol which are structurally related to paraoxon, were tested for their effects on interleukin 2 (IL2) production and responsiveness by rat splenocytes in vitro. Three of the four compounds inhibited mitogen-induced lymphocyte proliferation as well as IL2 production and responsiveness. However, phenyl saligenin cyclic phosphate produced maximal inhibition at a much lower concentration (0.5 μM) than p-nitrophenol (200 μM) or paraoxon (200 μM). Phenol was not inhibitory at any concentration tested (up to 250 μM). Since the production of and response to IL2 are key events in immune responses, compounds which suppress these events can be identified as potential suppressors of host resistance to disease.

Modulation of genotoxic and cytotoxic effects of aromatic amines in monolayers of rat hepatocytes.

Cultured rat hepatocytes exposed to 2-acetylaminofluorene (AAF), 2-aminofluorene (AF) or N-hydroxy-2-acetylaminofluorene (N-OH-AFF) for 3 hrs resulted in an increase in DNA repair measured as unscheduled DNA synthesis, with N-OH-AAF greater than AAF greater than AF. Cytotoxic effects were only seen with N-OH-AAF above 10(-6) M. alpha-Naphthoflavone increased the unscheduled DNA synthesis and cytotoxic effects of N-OH-AAF, whereas it decreased DNA repair and the covalent binding of AAF to cellular proteins. In contrast, very little effects of paraoxon were seen on the repair synthesis elicited by AAF, AF or N-OH-AAF. The addition of ascorbate reduced the covalent binding of AAF, the DNA repair synthesis caused by AAF and N-OH-AAF, and the cytotoxic effects of N-OH-AAF. The addition of pentachlorophenol or salicylamide all resulted in similar effects as ascorbate, through reduction of sulfation. Galactosamine, an inhibitor of glucuronidation, and the nucleophile GSH caused no or only minor effects of the activation of AAF, AF or N-OH-AAF as judged from the endpoints tested. These results are consistent with an arylnitrenium ion, a sulfate ester or a free radical as the arylamine metabolite causing cellular DNA damage, whereas the sulfate ester or a radical intermediate may be responsible for the cytotoxic effects of N-OH-AAF.

Effect of paraoxon on the level of cerebral cholinesterase activity, iv vitro and in vivo, in diabetic animals

Effect of paraoxon on the level of cerebral cholinesterase activity, iv vitro and in vivo, in diabetic animals

Effects of paraoxon and fenitrooxon on crustacean muscle membrane

1. The action of organophosphates such as paraoxon, methylparaoxon, fenitrooxon and ethyl-fenitrooxon was studied using neuromuscular junctions of lobster. 2. Paraoxon and methylparaoxon (1 mM) had no effect on presynaptic nerve spikes but produced depolarization in the muscle membrane of the lobster. 3. The cause of this depolarization is considered to be the activation of divalent cations, e.g. calcium ion, and not monovalent cations. 4. Fenitrooxon or ethylfenitrooxon, which are structurally similar to paraoxon, had little effect on muscle membrane spikes. 5. Paraoxon and methylparaoxon producing depolarizations, and the consequent contraction of the muscle membrane, might be responsible for the acute toxicity which is absent in fenitrooxon and ethylfenitrooxon.

THE PRESSOR EFFECTS OF PARAOXON IN THE PITHED RAT

1 In pithed rats paraoxon 825 microgram/kg induced a short-lasting pressor effect. Lower doses of the drug were ineffective. 2 The pressor effect was prevented by N-methylatropine, dexetimide and alpha-receptor blocking agents but not by mecamylamine. 3 When blood pressure of pithed rats was elevated either by the continuous infusion of vasopressin or by electrical stimulation of the pithing rod, both 275 and 825 microgram/kg paraoxon induced further pressor effects. The effectiveness of various receptor blocking agents was similar to that observed in pithed rats without vasopressin. 4 It is concluded that the pressor effect of paraoxon is mediated by ganglionic muscarinic receptors. Stimulation of these receptors by accumulated acetylcholine results in an increase in postganglionic sympathetic activity and causes pressor effects. 5 The peripheral action of paraoxon is compared with its action in intact anaesthetized animals.

The central effects of paraoxon on blood pressure of rabbits after intravenous administration or infusion via a vertebral artery.

In the present study paraoxon (28-825 micrograms/kg, i.v.) was administered into rabbits treated with N-methylatropine (1 mg/kg). In urethane-anaesthetized rabbits pressor and depressor effects were observed. In pentobarbitone-anaesthetized rabbits, however, only depresssor effects were noticed. Dexetimide (0.25-0.5 mg/kg) prevented the pressor and depressor action of paraoxon. Furthermore, paraoxon (83-825 micrograms) was infused into a vertebral artery of pentobarbitone-anaesthetized animals. After administration via this route the depressor effect was similar to i.v. administration. 30 min after administration of paraoxon acetylcholinesterase activity was determined in the medulla oblongata, the pons and the hypothalamus. The enzyme has to be inhibited almost completely to induce an effect on blood pressure. It is concluded that the vascular effects of paraoxon seem to be mediated by a mechanism involving stimulation of central muscarinic receptors, probably by accumulated acetylcholine, which in turn induces a decrease in blood pressure.

Inhibition of efferent sympathetic nerve activity by centrally administered paraoxon in the cat

We recently found that central administration of the cholinesterase inhibitor paraoxon lowered blood pressure substantially. It was postulated that the decrease in pressure was mediated by a reduction of sympathetic outflo. In the present study, efferent splanchnic nerve activity in anaesthesized and paralysed cats was recorded, and quantified by measuring the variance of signal amplitude. After administration of 8μg paraoxon into the vertebral arteries, blood pressure and splanchnic nerve activity decreased simulataneously. A mean fall of 46 ± 6% and 45 ± 13% ( X ± S.E.M.) respectively was reached within 12 min and was maintained during the perio studied (30 min). When the effect of paraoxon was antagonized by dexetimide, both blood pressure and splanchnic nerve activity returned to control values. Since previous work has shown that the depressor action could not be prevented by efferent vagal blockade it seems likely that the fall in blood pressure after paraoxon was mainly caused by a decreased symphathetic outflow. In addition, we varied the amplifier band width in recording splanchnic nerve activity. The measurement of frequencies between 10 and 225 Hz appeared to be sufficient for studying the change in activity after paraoxon.

Central effects of paraoxon on haemodynamics in the cat.

Application of paraoxon into the left vertebral artery (8--80 micrograms) or both the left and right vertebral artery (4--8 micrograms) of the anaesthetized cat evoked dose-dependent depressor effects, whereas heart rate was not influenced significantly. Also after systemic administration of paraoxon (150--825 micrograms . kg-1), while peripheral muscarinic receptors were blocked, depressor effects were still observed. Dose-response curves for the depressor response to paraoxon were established. Infusion of low doses of dexetimide via the vertebral artery prevented the hypotensive action of paraoxon. The distribution of this antimuscarinic drug in the brain was investigated. The depressor effect of paraoxon can be attributed to both a decrease in peripheral resistance and cardiac output. Decerebration and midcollicular transection were carried out in order to elucidate the site and mechanism of action. The depressor effect of paraoxon seems to be mediated by a central mechanism of action located within the lower brain stem. It is concluded that stimulation of muscarinic receptors in the pontomedullary region gives rise to the observed changes in haemodynamic parameters. Muscarinic receptors in the hypothalamus seem to be of minor importance for the hypotensive action of paraoxon.

Regional blood flow determinations in the rat during paraoxon-poisoning and treatment with atropine and obidoxime

Perfusion rates of various organs were determined for 4 different at populations subjected, respectively, to: (a) 15 min infusion of paraoxon (150 μg/kg i.v.); (b) 15 min infusion of paraoxon and the injection of 1.0 mg/kg atropinel; (c) 15 paraoxon infusion and the injection of 10.0 mg/kg obidoxine; and (d) none of these substances. Atropine and obidoxine were both injected 10 min after the beginning of the paraoxon infusion. The perfusion rates were determined by means of the microspheres technique, which allows the simultaneous measurement of different perfusion rates. The paraoxon-treated animals revealed a significantly lower perfusion rate of the kidney, skeletal muscle, skin and spleen in comparison to the unpoisoned control group. The flow rates in heart, stomach, small intestine, liver (arterial) and brain changed only slightly. The injection of atropine caused the perfusion rates of heart, kidney, intestine and spleen to be greater than under paraoxon alone. Obidoxime, furthermore, induced significant increases in the perfusion rates of all 9 organs studied. Indeed, for several organs, the flow rates under atropine and obidoxime were greater than those of the controls. It is suggested that the effects of paraoxon, atropine and obidoxime on the organ flow rates are due to different influences on peripheral and central muscarinic and nicotinic receptors and to changes in the depth of respiration.

Differential effect of a microsomal deacetylase inhibitor on the mutagenicity in Salmonella typhimurium of 2-acetylaminofluorene by liver homogenates of guinea pigs, mice and rats

The effect of paraoxon, a microsomal deacetylase inhibitor, on the mutant genicity of 2-acetylaminofluorene (AAF) by liver homogenates was compared between the AAF carcinogenesis-resistant guinea pigs and the susceptible mice and rats. The mutagenicity of AAF was mostly abolished by paraoxon, not only in the 3 kinds of untreated animals but also in guinea pigs treated with a combination of phenobarbital and 5,6-benzoflavone, whereas about 50% of the mutagenicity was resistant to paraoxon in treated mice and rats. We suggest that microsomal deacetylase activity is crucially involved in the mutagenic activation of AAF by guinea pig liver homogenates, while the enzyme activity other than the deacetylase activity is also important in the activation by liver homogenates from treated mice or rats.

Effects of paraoxon on axoplasmic transport of cholinesterase in rat sciatic nerve

Paraoxon, an irreversible inhibitor of organophosphorous cholinesterase, produces necrosis in innervated skeletal muscle. Because fast axoplasmic transport of protein has been suggested as a vehicle for maintenance of normal muscle properties, it is possible that it is involved in the muscle-damaging effect of the drug. To clarify this role, we have examined the effect of this drug on cholinesterase activity and fast axoplasmic transport of enzyme by measuring enzyme accumulation proximal to ligations of rat sciatic nerve. The results show that, in normal animals, fast axoplasmic transport of a small amount of available enzyme occurs at approximately 400 mm/day. Paraoxon treatment causes a significant reduction of enzyme activity in both muscle and nerve. The return of enzyme activity in muscle is rapid and continuous, whereas in nerve only a small recovery is seen during the first 2 hr after treatment. This difference in recovery of enzyme activity suggests that return of axonal enzyme occurs via somal synthesis and axoplasmic transport. The data also suggest that, although paraoxon is an effective inhibitor of cholinesterase, it does no alter fast axoplasmic transport of enzyme.

Effect of paraoxon on cholinesterase activity in certain brain regions of diabetic rats

The effect of paraoxon on the acetylcholinesterase activity of the cerebral cortex and corpus striatum of normal and diabetic rats has been determined. Paraoxon produced significantly less inhibition of cholinesterase activity in diabetic than in normal animals. The administration of insulin along with paraoxon restored the cholinesterase-inhibiting effect to the normal level. Paraoxon in both normal and diabetic rats produced a relatively greater degree of inhibition of cholinesterase activity in the striatum than in the cerebral cortex.