Studies on the Mode of Action of Organophosphorus Compounds

In order to further elucidate the mechanism of metabolic difference between sumithion and methylparathion, distribution of sumithion and methylparathion into several tissues, activation, that is, conversion into more toxic oxygen analogs, and degradation into non-toxic compounds were examined in vivo following the intravenous administration of the phosphorothioates to Guinea pigs and white rats. Sumioxon and methylparaoxon were detected in all tissues tested, among which lung and liver were richest in them. More sumioxon than methylparaoxon was found. Chese organophosphorus compounds were found to be decomposed to non-toxic desmethyl compounds and dimethyl phosphorothioic acid mainly in liver and kidney. From these results it seems improbable that the lower toxicity of sumithion than that of methylparathion results from the different in their rate of metabolism.     

Studies on the Mode of Action of Organophosphorus Compounds

For the purpose of distinguihsing sumithion from methylparathion in the mammalian metabolism, phosphorus³² labeled compounds were administered to Guinea pig and white rat. Both compounds were found to be absorbed readily, and phosphorus containing metabolites excreted chiefly into urine. By chromatographic separation and identification of the metabolites, the decomposition of sumithion was observed to proceed presumably more easily than methylparathion. From these results lower toxicity of the former toward mammals than the latter was discussed. In addition, compounds remaining in rice plant and German cockroach were also analysed.     

Metrifonate and dichlorvos: Effects of a single oral administration on cholinesterase activity in rat brain and blood

Cholinesterase activities in rat forebrain, erythrocytes, and plasma were assessed after a single oral administration of metrifonate or dichlorvos. In 3-month-old rats, the dichlorvos (10 mg/kg p.o.)-induced inhibition of cholinesterase reached its peak in brain after 15–45 min and after 10–30 min in erythrocytes and plasma. Cholinesterase activity recovered rapidly after the peak of inhibition, but did not reach control values in brain and erythrocytes within 24 h after drug administration. The recovery of plasma cholinesterase activity, in contrast, was already complete 12 h after dichlorvos treatment. Metrifonate (100 mg/kg p.o.) had qualitatively similar inhibition kinetics as dichlorvos, albeit with a slightly delayed onset. Peak values were attained 45–60 min (brain) and 20–45 min (blood), after drug administration. Apparently complete recovery of cholinesterase activity was noted in both tissues 24 h after treatment. The dose-dependence of drug-induced inhibition of cholinesterase in rat blood and brain was determined at the time of maximal inhibition, i.e., 30 min after dichlorvos treatment and 45 min after metrifonate treatment. The oral ED₅₀ values obtained for dichlorvos were 8 mg/kg for brain and 6 mg/kg for both erythrocyte and plasma cholinesterase. The corresponding oral ED₅₀ values for metrifonate were 10 to 15 times higher, i.e., 90 mg/kg in brain and 80 mg/kg in erythrocytes and plasma. In rats deprived of food for 18 h before drug treatment, the corresponding ED₅₀ values for metrifonate were 60 and 45 mg/kg, respectively, indicating an about two-fold higher sensitivity of fasted rats to metrifonate-induced cholinesterase inhibition compared to non-fasted rats. Compared to 3-month-old rats, 19-month-old rats showed a higher sensitivity towards metrifonate and dichlorvos. At the time of maximal inhibition, there was a strong correlation between the degree of cholinesterase inhibition in brain and blood. These results demonstrate that single oral administration of metrifonate and dichlorvos induces an inhibition of blood and brain cholinesterase in the conscious rat in a dose-dependent and apparently fully reversible manner. While the efficiency of a given dose of inhibitor may vary with the satiety status or age of the animal, the extent of brain ChE inhibition can be estimated from the level of blood ChE activity.     

A comparison of cholinesterase activity after intravenous, oral or dermal administration of methyl parathion

Time-dependent changes in blood cholinesterase activity caused by single intravenous, oral or dermal administration of methyl parathion to adult female rats were defined. Intravenous and oral administration of 2.5 mg/kg methyl parathion resulted in rapid (<60 min) decreases in cholinesterase activity which recovered fully in vivo within 30-48 h. In contrast, spontaneous reactivation of cholinesterase in vitro was complete within 6 h at 37 degrees C. Dermal administration of methyl parathion caused dose-dependent inhibition of cholinesterase activity which developed slowly (> or =6 h) and was prolonged (> or =48 h). Time- and route-dependent effects of methyl parathion on cholinesterase activity in brain and other tissues generally paralleled its effects on activity in blood. In conclusion, pharmacodynamics of methyl parathion differ substantially with route of exposure. Recovery of cholinesterase in vivo after intravenous or oral exposure may partially reflect spontaneous reactivation and suggests a rapid clearance of methyl parathion or its active metabolite methyl paraoxon. The more gradual and prolonged inhibition of cholinesterase caused by dermal administration is consistent with disposition of methyl parathion at a site from which it or methyl paraoxon is only slowly distributed. Thus, dermal exposure to methyl parathion may pose the greatest risk for long-term adverse effects.     

Effects of diethyldithiocarbamate on the acute toxicity and the acetylcholinesterase inhibition by methylparathion in mice]

Acute toxicity of methylparathion male mice is reduced by DET in dose-range from 50 to 300 mg/kg. Mean survival time in LD100 range of methylparathion is significantly prolonged. Inhibition of AChE in vivo is reduced, too. The observed dose-effect-relations show an approx. 10-fold increase in equi-effective doses after DET-pretreatment. DET has no protective effect both in vivo and in vitro, if methylparathion is added to the brain homogenate of mice. As probable mechanism of action a change in methylparathion-metabolism induced by DET in the system of mixed functional oxidases is discussed.     

The relationships between brain, serum, and whole blood ChE activity in the wood mouse (Apodemus sylvaticus) and the common shrew (Sorex araneus) after acute sublethal exposure to dimethoate

Inhibition of cholinesterase (ChE) activity produced by a single acute intraperitoneal administration of dimethoate was studied in the wood mouse, Apodemus sylvaticus, and the common shrew, Sorex araneus, under laboratory conditions. ChE values from serum and whole blood were compared with those obtained from brain in order to obtain a non-destructive tool for predicting the severity of brain acetylcholinesterase (AChE) inhibition. In addition, serum and brain inhibition following oral exposure to dimethoate was also measured in the wood mouse. Normal ChE activity was higher in the brain and whole blood of the shrews than in wood mice. There was no difference between species in serum ChE activity. Exposure to dimethoate caused a dose-dependent reduction in ChE activity and there was a significant recovery in activity with increasing time after administration. In both species, serum and whole blood were more sensitive than brain for revealing organophosphate-induced ChE inhibition and serum was more sensitive than whole blood. Statistically significant relationships were defined between whole blood and brain ChE activity and between serum and brain ChE activity. Compared with serum, whole blood ChE activity was the more accurate predictor of brain AChE levels. The relationships between brain and serum ChE activity did not appear to be affected by the route of administration of the pesticide.     

The relationships between brain,serum, and whole blood ChE activity in the wood mouse (Apodemus sylvaticus) and the common shrew (Sorex araneus) after acute sublethal exposure to dimethoate

Abstract

Inhibition of cholinesterase (ChE) activity produced by a single acute intraperitoneal administration of dimethoate was studied in the wood mouse, Apodemus sylvaticus, and the common shrew, Sorex araneus, under laboratory conditions. ChE values from serum and whole blood were compared with those obtained from brain in order to obtain a non-destructive tool for predicting the severity of brain acetylcholinesterase (AChE) inhibition. In addition, serum and brain inhibition following oral exposure to dimethoate was also measured in the wood mouse. Normal ChE activity was higher in the brain and whole blood of the shrews than in wood mice. There was no difference between species in serum ChE activity. Exposure to dimethoate caused a dose-dependent reduction in ChE activity and there was a significant recovery in activity with increasing time after administration. In both species, serum and whole blood were more sensitive than brain for revealing organophosphate-induced ChE inhibition and serum was more sensitive than whole blood. Statistically significant relationships were defined between whole blood and brain ChE activity and between serum and brain ChE activity. Compared with serum, whole blood ChE activity was the more accurate predictor of brain AChE levels. The relationships between brain and serum ChE activity did not appear to be affected by the route of administration of the pesticide.     

Interaction between various cholinesterases and reversible inhibitors of polymethylene-bis-(trimethylammonium) diiodide series

It is established that derivatives of polymethylene bistrimethylammonium (CH3)3N+(CH2)nN+(CH3)3 (n = 4-10) are reversible competitive and mixed action inhibitors with respect to acetylcholinesterase of human erythrocytes, butyryl cholinesterase of horse blood serum, cholinesterase of frog brain and Todarodes pacificus optical ganglion. In case of mammals and frog cholinesterase the inhibitors efficiency rises with n, but the activity of the Todarodes pacificus cholinesterase less sensitive of the inhibitors is characterized by a "step" dependence on the length of the polymethylene chain of the inhibitor molecule. Studies in sensitivity of cholinesterases to this type of inhibitors revealed differences between enzymes of the same type in different animals.     

The effect of heptyl-physostigmine,a new cholinesterase inhibitor,on the central cholinergic system of the rat

Heptyl-physostigmine (Heptyl-Phy; MF-201) is a new carbamate derivative of physostigmine (Phy) with greater lipophilicity and longer inhibitory action on cholinesterase (ChE) activity than the parent compound. Following single dose administration of 5 mg/kg heptyl-Phy i.m., maximal whole brain acetylcholinesterase (AChE) inhibition (82%) if reached at 60 min. Inhibition of plasma BuChE butyrylcholinesterase (BuChE) remains close to the steady state level (60%) between 120 and 360 min. At 360 min, whole brain AChE activity is still 67% inhibited compared to controls. Inhibition of AChE activity displays brain regional differences which are more significant at 360 min. At this time point, AChe activity in cerebellum is only 40% inhibited while frontal cortex and medial septum are still 80% inhibited. Increases in acetycholine (ACh) levels also show regional differences, however, there is no direct relationship between AChE inhibition and ACh increase. The electrically evoked [³H]ACh release in cortical slices was inhibited only by the highest concentration of heptyl-Phy tested (10^–4M). At this concentration ChE activity was 97% inhibited in vitro. In conclusion, our results demonstrate that heptyl-Phy compares favorably to other reversible cholinesterase inhibitors (ChEI), particularly to Phy as far as producing a more long-lasting inhibition of AChE and a more prolonged increase of ACh in brain with less severe side effects. Therefore, it represents an interesting candidate for cholinomimetic therapy of Alzheimer disease (AD).Dept. of Pharmacology, Shanghai Institute of Materia Medica Chinese Academy of Sciences Shanghai 20031 China.Special issue dedicated to Dr. Paola S. Timiras     

Melatonin Protects Against Diazinon-Induced Neurobehavioral Changes in Rats

Diazinon is an organophosphorous pesticide with a prominent toxicity on many body organs. Multiple mechanisms contribute to diazinon-induced deleterious effects. Inhibition of acetyl-cholinesterase, cholinergic hyperstimulation, and formation of reactive oxygen species may play a role. On the other hand, melatonin is a pineal hormone with a well-known potent antioxidant activity and a remarkable modulatory effect on many behavioral processes. The present study revealed that oral diazinon administration (25 mg/kg) increased anxiety behavior in rats subjected to elevated plus maze and open-field tests possibly via the induction of changes in brain monoamines levels (dopamine, norepinephrine, and serotonin). Additionally, brain lipid peroxides measured as malondialdehyde (MDA) and tumor necrosis factor alpha (TNF-α) levels were elevated, while the activity of brain glutathione peroxidase enzyme was reduced by diazinon. Co-administration of oral melatonin (10 mg/kg) significantly attenuated the anxiogenic activity of diazinon, rebalanced brain monoamines levels, decreased brain MDA and TNF-α levels, and increased the activity of brain glutathione peroxidase enzyme.     

Identical reactivity of brain and erythrocyte cholinesterases of some mammals

The paper deals with a comparative study of various aspects of reactivity (substrate and inhibitor specificity, sensitivity to action of hydrophobic organophosphorus inhibitors, capability for reactivation) of cholinesterase preparations from brain, erythrocytes, and serum of a group of mammals (human, rabbit, rat, cattle, dog, and cat). It has turned out that at preservation of the species and tissue specificity, catalytic properties of the brain and erythrocyte cholinesterases of the same animal species are very close to each other.     

Oxidation of cholinesterase-inhibiting pesticides: A simple experiment to illustrate the role of bioactivation in the toxicity of chemicals

A simple undergraduate laboratory experiment that can be used in Biochemistry and Toxicology courses to illustrate the importance of metabolic reactions in the toxicity of chemical substances is reported. It involves the experimental confirmation that oxidized phosphorothionate esters, commonly used as insecticides, are stronger cholinesterase inhibitors and therefore exhibit higher toxicity than do their sulphur analogs starting from which the first are formed by in vivo oxidative desulphuration. Two separated aliquots of a bovine blood sample are incubated with parathion and paraoxon, its oxygen analog, and compared for cholinesterase activity with "normal" blood. Previously, a standard sample of paraoxon was obtained by oxidation of the thiono group of parathion with bromine vapour by reaction TLC. The comparison of the inhibitory capacity of both compounds is made by a colorimetric procedure using acetylthiocholine as substrate of the enzyme and 5,5'-dithiobis-(2-nitrobenzoic acid) as chromogen.     

Comparative Study of Reversible Organofluorine Ammonium Inhibitors of Cholinesterases of Different Animals

A group of organofluorine ammonium compounds, trimethyltrifluoromethylammonium, diethylmethyltrifluoromethylammonium, hexa(difluoromethylene)-bis(trimethylammonium), their non-substituted analogs as well as bis-onium organosilicone, phenyliodonium, and triphenylphosphonium derivatives were tested as reversible inhibitors of acetylcholinesterase of human erythrocytes, butyrylcholinesterase of horse blood serum, cholinesterase of brain of the frog Rana temporaria and cholinesterases of optic ganglion of the Pacific squid Todarodes pacificus. By the method of molecular mechanics, differences were revealed in conformational mobility of interonium chain and in geometric parameters of the studied compounds. It was shown that introduction of fluorine atoms into the inhibitor molecule affected only their interaction with the Pacific squid cholinesterase. It was possible to separate effects of the onium atom nature and of the interonium chain structure in the inhibitor molecule on the anticholinesterase potency.     

Effect of pesticide bendiocarbamate on distribution of acetylcholine- and butyrylcholine-positive nerves in rabbit's thymus

Many pesticides used in agriculture have a negative effect on organisms. The group of hazardous pesticides includes the cholinesterase inhibitor bendiocarbamate. According to literature, bendiocarbamate has relatively low toxicity in mammals and vertebrates in general, since it does accumulate in their tissues and the cholinesterase activity returns to norm within 24 h after acute exposure. The present study focused on the influence of bendiocarbamate on rabbit thymus after its administration at a dose of 5 mg/kg body weight (BW) for the period of 3 months. The thymus was observed for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)-positive nerve fibers visualized by histochemical methods. Microscopic findings of BuChE-positive nerve fibers show the same density and the topography by the experimental and the control animals. On the other hand, AChE-positive nerve fibers in experimental animals after administration of bendiocarbamate is only poorly identified, suggesting that bendiocarbamate inhibits AChE but not BuChE.     

Analogs that compete for antigen binding to an arsonate-reactive T-cell clone inhibit the functional response to arsonate

We have tested several structurally related haptens, conjugated to ovalbumin, for their effect on activation of an inducer T-cell clone reactive to the pazobenzenearsonate (arsonate) hapten. Low concentrations of some analogs inhibited DNA synthesis and lympkokine production by the clone in response to arsanylated antigen, but not in response to the lectin concanavalin A. Inhibition was specific for this clone, since the response of clones reactive to other antigens was not blocked. Inhibition may result from competition of these analogs with arsonate at a site on the T cell. The effectiveness of blocking by arsonate analogs parallels their ability to bind to a previously described arsonate-binding site on the clone (Rao et al., accompanying paper). We suggest that the binding and blocking assays detect the same physiological arsonate-recognition site on the clone, and hence that the cell-surface arsonatebinding sites we have described mediate its physiological response to antigen.     

Modification by physostigmine of the cardiovascular responses to intracerebroventricular administration of 5-hydroxytryptamine in rats

In rats the effect of inhibition of the brain cholinesterase activity on the pressor and heart rate responses to 5-hydroxytryptamine (5-HT), administered into the lateral cerebral ventricle (l.c.v.) was examined. After administration of physostigmine (twice in a small dose of 2.5 micrograms l.c.v., 20 and 15 min before the second injection of 5-HT), the pressor effect of 5-HT (5 micrograms) was strongly reduced or almost abolished, its pure tachycardia was reduced or reversed into a bradycardia and its pure bradycardia was diminished or reversed into a tachycardia. The type of the cardiovascular response to ACh (5 micrograms l.c.v., 20 min after the second administration of 5-HT) indicates that the modification of the cardiovascular response to 5-HT was accompanied by inhibition of the brain cholinesterase activity. Thus, it seems that a functionally competent cholinesterase in the brain is necessary for the generation of the 5-HT-induced pressor response. The present experiments provide further evidence that there is a cholinergic link in the pathway by which serotonergic mechanisms in the preoptic-anterior hypothalamic area rise blood pressure and support the idea that the same link exists in the pathway(s) mediating the heart rate responses to intracerebroventricular administration of 5-HT.     

Changes induced by malathion, methylparathion and parathion on membrane lipid physicochemical properties correlate with their toxicity

Perturbations induced by malathion, methylparathion and parathion on the physicochemical properties of dipalmitoylphosphatidylcholine (DPPC) were studied by fluorescence anisotropy of DPH and DPH-PA and by differential scanning calorimetry (DSC). Methylparathion and parathion (50 microM) increased the fluorescence anisotropy evaluated by DPH-PA and DPH, either in gel or in the fluid phase of DPPC bilayers, but mainly in the fluid phase. Parathion is more effective than methylparathion. On the other hand, malathion had almost no effect. All the three xenobiotics displaced the phase transition midpoint to lower temperature values and broadened the phase transition profile of DPPC, the effectiveness following the sequence: parathion>methylparathion>malathion. A shifting and broadening of the phase transition was also observed by DSC. Furthermore, at methylparathion/lipid molar ratio of 1/2 and at parathion/lipid molar ratio of 1/7, the DSC thermograms displayed a shoulder in the main peak, in the low temperature side, suggesting coexistence of phases. For higher ratios, the phase transition profile becomes sharp as the control transition, but the midpoint is shifted to the previous shoulder position. Conversely to methylparathion and parathion, malathion did not promote phase separation. The overall data from fluorescence anisotropy and calorimetry indicate that the degree of effect of the insecticides on the physicochemical membrane properties correlates with toxicity to mammals. Therefore, the in vivo effects of organophosphorus compounds may be in part related with their ability to perturb the phospholipid bilayer structure, whose integrity is essential for normal cell function.     

Synthesis and antinociceptive effects of endomorphin-1 analogs with C-terminal linked by oligoarginine

Endomorphins (EMs) cannot be delivered into the central nervous system (CNS) in sufficient quantity to elicit antinociception when given systemically because they are severely restricted by the blood-brain barrier (BBB). In the present study, we investigated herein a series of EM-1 analogs with C-terminal linked by oligoarginine in order to improve the brain delivery and antinociception after systemic administration. Indeed, all these analogs decreased the opioid receptor affinity and in vitro pharmacological activity. Moreover, analogs 4, 7-9 produced a less potent antinociceptive activity after intracerebroventricular (i.c.v.) administration, with the ED₅₀ values about 11- to 13-fold lower potencies than that of EM-1. Nevertheless, our results revealed that EM-1 failed to induce any significant antinociception at a dose of 50 μmol/kg after subcutaneous (s.c.) administration, whereas equimolar dose of these four analogs produced a little low but significant antinociceptive effects. Naloxone (10 nmol/kg, i.c.v.) significantly blocked the antinociceptive effects, indicating an opioid and central mechanism. These results demonstrated that C-terminal of EM-1 linked to oligoarginine improved the brain delivery, eliciting potent antinociception following peripheral administration.     

Anticholinesterase effect and toxicity of bis(trichloromethyl) sulfone (N-1386 Biocide) in rats

The oral LD50 for bis(trichloromethyl) sulfone (N-1386 Biocide) in male rats was 691 mg/kg. Deaths occurred 1-5 days after treatment and signs of toxicity suggestive of an anticholinesterase effect were noted. However, neither plasma cholinesterase nor brain acetylcholinesterase was inhibited 2, 4 or 24 hours after a single, oral dose of 500 mg/kg. Atropine (300 mg/kg, s.c.) or scopolamine (670 mg/kg, s.c.) pretreatments did not protect against the acute lethality of bis(trichloromethyl) sulfone although signs of toxicity were alleviated by both pretreatments. Bis(trichloromethyl) sulfone produced in vitro inhibition of rat plasma cholinesterase and brain acetylcholinesterase. The inhibition was competitive in brain. IC50's for these 2 enzymes were 8 microM in plasma and 25 microM in brain. In summary, bis(trichloromethyl) sulfone produced in vitro cholinesterase inhibition not demonstrated in vivo. Doses of anticholinergic compounds that ameliorated many toxic signs did not protect against lethality produced by bis(trichloromethyl) sulfone.     

Intestinal metabolism and absorption of cholecystokinin analogs in rats

Intestinal metabolism and poor permeability were known to be major barriers for oral absorption of large peptide drugs. Dimensionless wall permeability values of C-terminal octa- and tetra-peptides cholecystokinin analogs (CCK8 and CCK4) were estimated and found out to be greater than 1, suggesting no permeability-limited absorption for CCK analogs. Thus, a strategy employing enzyme inhibitors and a specific delivery site to improve the absorption was developed and tested with CCK8, followed by identification of metabolites of the analogs and their participating enzymes in rabbit brush-border membrane vesicles. Thiorphan and amastatin, a specific enzyme inhibitor for enkephalinase and aminopeptidase, respectively, in pH 4 buffer solution were coadministered with CCK8 to the ileum in fistulated rats. The absolute bioavailability (F) of CCK8 was 5.4% and increased to 19% in the presence of the enzyme inhibitors, while the F values following oral administration were close to zero. These results indicate that peptide oral delivery is possible.