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Role of detoxication pathways in acute toxicity levels of phosphorothionate insecticides in the rat
Abstract
Phosphorothionate insecticides and their active oxon metabolites can be detoxified by a variety of hepatic mechanisms. Cytochrome P450-mediated dearylation activity was higher in males than in females. While dearylation was induced by phenobarbital in both sexes, it was induced by beta-naphthoflavone in females only. Detoxication of oxons in the presence of EDTA was inducible by phenobarbital, was higher in males than in females, and paralleled aliesterase activity. In vitro Ca(++)-dependent A-esterase-mediated hydrolysis of chlorpyrifos-oxon but not of paraoxon occurred at biologically relevant nM concentrations. This hydrolysis was also inducible by phenobarbital. Glutathione-mediated conjugation did not appear to be relevant to the disposition of the phosphorothionates studied here. Hepatic detoxication via dearylation, aliesterase phosphorylation and A-esterase-mediated hydrolysis (for some organophosphates) all appear to be relevant reactions in the attenuation of acute toxicity.
... Several studies on desulfuration and dearylation of phosphorothionates by hepatic microsomes have been carried out in mammals (Levi et al., 1988;Forsyth and Chambers, 1989;Chambers et al., 1994); these activities are inducible in rats by phenobarbitol (Ma and Chambers, 1995) and b-naphthoflavone (Chambers et al., 1994). There are reports of phosphorothionate metabolism in fish (Potter and O'Brien, 1964;Murphy, 1966;Benke et al., 1974;Wallace and Dargan, 1987;Boone and Chambers, 1997); however, hepatic microsomal metabolism has only been reported by Boone and Chambers (1997). ...
... Several studies on desulfuration and dearylation of phosphorothionates by hepatic microsomes have been carried out in mammals (Levi et al., 1988;Forsyth and Chambers, 1989;Chambers et al., 1994); these activities are inducible in rats by phenobarbitol (Ma and Chambers, 1995) and b-naphthoflavone (Chambers et al., 1994). There are reports of phosphorothionate metabolism in fish (Potter and O'Brien, 1964;Murphy, 1966;Benke et al., 1974;Wallace and Dargan, 1987;Boone and Chambers, 1997); however, hepatic microsomal metabolism has only been reported by Boone and Chambers (1997). ...
... Dearylation was quantified by a modification of the method described by Chambers et al. (1994). Liver microsomes, 100 mg wet weight equivalent, were incubated in 100 mM Tris-HCl+ 5 mM MgCl 2 (pH 7.4) buffer at 30°C for 15 min in the presence or absence of 50 ml NADPH-generating system consisting of 100 mM glucose-6-phosphate, 10 mM NADP + and 1.6 U glucose-6-phosphate dehydrogenase; final volume was 1 ml for Cp and 500 ml for Pth. ...
Channel catfish were treated intraperitoneally with 100 mg Aroclor 1254/kg body weight and sacrificed at 96 h to observe the effects of this cytochrome P450 1A (CYP1A) inducer on chlorpyrifos and parathion metabolism. In the initial experiment, hepatic microsomal ethoxyresorufin-O-deethylase (EROD) activity of the Aroclor-treated fish was significantly induced but no effects on desulfuration or dearylation of chlorpyrifos or parathion were evident. In the second experiment, Aroclor 1254 did not alter total hepatic microsomal P450s content, but significantly induced hepatic EROD and CYP1A. There were no evident effects to other hepatic CYP isoforms recognized by anti-trout CYP2K1, CYP2M1 and CYP3A27. These experiments indicate that Aroclor 1254 did not induce the P450s responsible for metabolism of the phosphorothionate insecticides.
... Some cytochrome P450 isomers are more proficient at dearylation than at oxidative desulfuration [37,38]. Chlorpyrifos induces the expression of isomers that are more active at dearylation (inactivation) than at desulfuration (activation) [37,39]. The levels of induced cytochrome P450 correlate with the amount of chlorpyrifos in circulation, so that higher levels of the detoxifying isomer are expected in response to higher levels of chlorpyrifos [33]. ...
... Consequently, CPO and CF were more quickly inactivated in the ES1−/− mice than in the ES1+/+ mice. Support for this hypothesis comes from a study in rats which found that treatment with chlorpyrifos induced 4-fold more dearylation than desulfuration, whereas treatment with parathion induced 4 to 8-fold more desulfuration than dearylation [39]. This hypothesis is expected to apply only to a limited range of chlorpyrifos or chlorpyrifos oxon doses. ...
... This suggests that the protective CYP orthologs are induced specifically by organochlorines and not by phosphorothioates in general. In support of this suggestion, a study in rats found that treatment with chlorpyrifos induced 4-fold more dearylation than desulfuration, whereas treatment with parathion induced 4 to 8-fold more desulfuration than dearylation [39]. ...
Mouse blood contains four esterases that detoxify organophosphorus compounds: carboxylesterase, butyrylcholinesterase, acetylcholinesterase, and paraoxonase-1. In contrast human blood contains the latter three enzymes but not carboxylesterase. Organophosphorus compound toxicity is due to inhibition of acetylcholinesterase. Symptoms of intoxication appear after approximately 50% of the acetylcholinesterase is inhibited. However, complete inhibition of carboxylesterase and butyrylcholinesterase has no known effect on an animal's well being. Paraoxonase hydrolyzes organophosphorus compounds and is not inhibited by them. Our goal was to determine the effect of plasma carboxylesterase deficiency on response to sublethal doses of 10 organophosphorus toxicants and one carbamate pesticide. Homozygous plasma carboxylesterase deficient ES1(-/-) mice and wild-type littermates were observed for toxic signs and changes in body temperature after treatment with a single sublethal dose of toxicant. Inhibition of plasma acetylcholinesterase, butyrylcholinesterase, and plasma carboxylesterase was measured. It was found that wild-type mice were protected from the toxicity of 12.5mg/kg parathion applied subcutaneously. However, both genotypes responded similarly to paraoxon, cresyl saligenin phosphate, diisopropylfluorophosphate, diazinon, dichlorvos, cyclosarin thiocholine, tabun thiocholine, and carbofuran. An unexpected result was the finding that transdermal application of chlorpyrifos at 100mg/kg and chlorpyrifos oxon at 14mg/kg was lethal to wild-type but not to ES1(-/-) mice, showing that with this organochlorine, the presence of carboxylesterase was harmful rather than protective. It was concluded that carboxylesterase in mouse plasma protects from high toxicity agents, but the amount of carboxylesterase in plasma is too low to protect from low toxicity compounds that require high doses to inhibit acetylcholinesterase.
... Both desulfuration and dearylation reactions occur through a common phosphooxythiiran intermediate and can be mediated by the same cytochrome P450 enzyme (Neal, 1980). Cytochrome P450 inducers, such as phenobarbital, have been reported to increase both the desulfuration and dearylation of the three phosphorothionate insecticides selected for study here (Alary and Brodeur, 1969;Chambers et al., 1994;Ma and Chambers, 1995;Sultatos, 1986Sultatos, , 1987Sultatos et al., 1984). Pretreatment with phenobarbital actually decreases the toxicity of these phosphorothionates Sultatos, 1986;Sultatos et al., 1984Sultatos et al., , 1987. ...
... Previous work has reported that pre-exposure to inducers of cytochrome P450 can provide some degree of protection against the toxicity of organophosphorus insecticides (Alary and Brodeur, 1969;Ball et al., 1954;Brodeur, 1967;Chambers et al., 1994;Harbison, 1975;Mourelle et al., 1986;Sultatos, 1986;Sultatos and Minor, 1987;Sultatos et al., 1984;Triolo and Coon, 1966a,b;Welch and Coon, 1964). Thus, it was not surprising that at least some similar results were obtained in this study with PCBs. ...
Exposure to polychlorinated biphenyls (PCBs) can alter the metabolism of organophosphorus (OP) insecticides. Female rats were fed vanilla wafers containing either 4 mg/kg/day of Aroclor 1254 (PCBtreated) or safflower oil (oil-treated) for 50 days. Rats were then injected, ip, with corn oil, parathion (P5S), methyl parathion (MP5S), chlorpyrifos (C5S), paraoxon (P5O), methyl paraoxon (MP5O), or chlorpyrifos-oxon (C5O). In the livers of rats treated with PCBs but not OP compounds, there was induction of desulfuration (activation) of P5S, MP5S, and C5S, but dearylation (detoxication) was induced only with P5S and MP5S. Hepatic Aesterase hydrolysis of all three oxons was induced. Cholinesterase (ChE) activity was determined in the medulla-pons, hippocampus, corpus striatum, cerebral cortex, skeletal muscle, lung, and heart at 2 and 24 h post exposure. With C5S, P5S, and MP5S, differences in brain ChE inhibition were observed a t2h( MP5S > P5S > C5S) but few differences were observed between oil- and PCB-treated rats. By 24 h, the level of brain ChE inhibition had increased with P5S and C5S but had decreased with MP5S. In rats exposed to P5S and C5S but not MP5S, ChE inhibition was lower in PCB-treated rats than in oil-treated rats. This suggests that pre-exposure to PCBs has a protective effect against the acute toxicity of P5S and C5S, but not MP5S. This protective effect does not appear to be related to the alteration of the metabolism of these compounds. The slower rate of ChE inhibition following P5S and C5S compared to MP5S suggests that the protection may be mediated by the PCB-induced increase in A-esterase activity. This protection appears to be related to the time between exposure and inhibition of ChE. With the oxons at 2 h, inhibition of ChE was substantial and no differences were present between the PCB- and oil-treated rats. Thus, the rapid rate of inhibition of ChE by the oxons does not afford time for the increase in A-esterase hydrolysis to effectively provide protection against inhibition of ChE. However, while no differences between oil- and PCB-treated rats were observed with MP5O by 24 h, PCB-treated rats exposed to P5O and C5O had lower ChE inhibition than did oil-treated rats with greater differences observed with P5O than
... CPS is bioactivated readily in vivo to its active metabolite, chlorpyrifos-oxon (CPO), a very potent anticholinesterase (Chambers et al., 1990). However, detoxication pathways in the liver and plasma, such as P450-mediated dearylation of the parent insecticide, carboxylesterase (CbxE; known also as aliesterase), phosphorylation by the oxon, and A-esterase-mediated hydrolysis of the oxon can degrade CPS or CPO, and thus reduce the in vivo toxicity of CPS (Chambers et al., 1990(Chambers et al., , 1994. Activity of these detoxication enzymes is lower in juveniles than in adults (Atterberry et al., 1997;Benke and Murphy, 1975;Mortensen et al., 1996), leaving the juvenile nervous system more vulnerable to attack by OP anticholinesterases. ...
... Similar findings were reported by others (Chanda et al., 1995;Chanda and Pope, 1996), in which pups cross-fostered to CPS-treated dams showed small but significant reductions in ChE activity. CPS can be detoxified by P450 dearylation, and its active metabolite, CPO, by A-esterase hydrolysis and CbxE phosphorylation (Chambers et al., 1994). CbxE phosphorylation (displayed as inhibition of activity) not only detoxifies oxon molecules but also is a sensitive marker for OP exposure. ...
The effects of repeated oral exposures to the organophosphorus insecticide chlorpyrifos (CPS) on brain muscarinic receptor densities, together with cholinesterase (ChE) activity, were studied in early postnatal rats. Initially, the effects on esterases from lactational exposure to CPS were investigated in young rats by administering CPS (100, 150, or 200 mglkg subcutaneously in corn oil) to dams 1 day postpartum, yielding a significant body burden of CPS in the dams for possible excretion in the milk. Brain ChE inhibition in pups was less severe than in dams, whereas liver carboxylesterase (CbxE) inhibition in pups was at the same level as in dams. Because of the limited brain ChE inhibition obtained following lactation, pups were exposed to CPS directly by gavage, using 3 dosing regimens to yield a dose response. The rats were gavaged with CPS in corn oil on alternate days from postnatal day (PND) 1 through PND 21. Rats in the low-dosage group received 11 treatments at 3 mg/kg, those in the medium-dosage group received 3 treatments at 3 mg/kg and 8 at 6 mg/kg, and those in the high dosage group received 3 treatments at 3 mglkg, 4 at 6 mg/kg, and 4 at 12 mg/kg. ChE activity in brain homogenates were inhibited significantly by 29% and 63% in the low- and high-dosage groups, respectively, on PND 22 and by 17% in the high dosage group on PND 40. Muscarinic receptor densities in brain synaptosomes were reduced using 3 H-N-methylscopolamine (NMS) and 3 H-quinuclidinyl benzilate (QNB) as ligands, with the effects more prominent from 3 H-NMS. Densities of both ligands recovered to the control level several days after terminating treatment. The results indicate that pups are apparently exposed to only limited amounts of chlorpyrifos and/or its oxon through the milk when dams are exposed to extremely high chlorpyrifos levels. In addition, repeated direct oral exposures of early postnatal rats to CPS will result in persistent brain ChE inhibition and will transiently reduce muscarinic receptor density.
... The in vitro PON1-mediated hydrolysis of high and low CPO concentrations was assessed using either a direct or indirect spectrophotometric method, respectively. The indirect method allows for quantitation of CPO hydrolysis using a lower, more environmentally relevant nanomolar concentration than the high micromolar concentration required by the direct assay for spectrophotometric quantification of the hydrolysis product (Chambers et al., 1994). The indirect assay measures the CPO remaining following PON1 hydrolysis by assessing the inhibition of an exogenous source of AChE by the residual CPO. ...
... The indirect assay of hydrolysis toward a low concentration of CPO was performed as described in Chambers et al. (1994) and Pond et al. (1996) with a few modifications. Human serum samples (10 μl serum/ml 0.1 M Tris-HCl buffer at pH 7.4) containing either 1 mM EDTA or 2 mM CaCl 2 were incubated with shaking at 37°C for 15 min. ...
The organophosphorus insecticide chlorpyrifos has been widely used. Its active metabolite chlorpyrifos-oxon (CPO) is a potent anticholinesterase and is detoxified by paraoxonase 1 (PON1). PON1 activity is influenced by numerous factors including a Q192R polymorphism. Using forty human blood samples bearing homozygous genotypes and either high or low activity phenotypes (as determined by high concentration assays of paraoxon and diazoxon hydrolysis) the serum PON1 hydrolysis of high (320μM) and low (178nM) CPO concentrations was assessed using direct or indirect spectrophotometric methods, respectively. PON1 activity at high CPO concentration reflected the phenotype and genotype differences; subjects with the high activity phenotype and homozygous for the PON1R192 alloform hydrolyzed significantly more CPO than subjects with the low activity phenotype and/or PON1Q192 alloform (high RR=11023±722, low RR=9467±798, high QQ=8809±672, low QQ=6030±1015μmol CPO hydrolyzed/min/L serum). However, PON1 hydrolysis of CPO at the lower, more environmentally relevant concentration showed no significant differences between the PON1192 genotypes and/or between high and low activity phenotypes (high RR=231±27, low RR=219±52, high QQ=193±59, low QQ=185±43nmol CPO/min/L serum). Low CPO concentrations were probably not saturating, so PON1 did not display maximal velocity and the PON1 genotype/phenotype might not influence the extent of metabolism at environmental exposures.
... It is well known that xenobiotics are enzymatically metabolised in the liver of fish. According to Chambers et al. (1994), phosphrothionate insecticides and their active metabolites can be detoxified by a variety of hepatic mechanisms. This includes P-450-mediated dearylation, phosphorylation of aliesterases (carboxylesterases), catalytic hydrolysis of oxon by A-esterases, glutathion (GSH)-dependent detoxification, etc. ...
... TPP is a known inhibitor of carboxyl esterase (Oppenoorth and Welling, 1976). Carboxyl esterase belongs to a group of hydrolytic enzymes, collectively called non-specific esterases, that are important in detoxification of OP compounds and synthetic pyrethroids (Dauterman, 1976;Kao et al., 1985;Chambers et al., 1994). Hence, the observed increase in acute toxicity of FNT to TPP pre-treated fish may be due to inability of the fish to detoxify FNT. ...
The acute toxicity of fenitrothion (FNT) alone and combined with piperonyl butoxide (PBO) or triphenyl phosphate (TPP), and their effect on liver esterase (LE ) activity of Gambusia holbrooki, Pseudorasobora parva and Oncorhynchus mykiss was studied in aquaria for 96h. The results indicated that fenitrothion is moderately toxic to the three species of fish tested, the toxicity being highest to O. mykiss followed by P. parva and G. holbrooki. Both FNT concentrations and the exposure time on the activity of LE and FNT (two way ANOVA P<0.05) caused more inhibition in G. holbrooki than P. parva . Pretreating fish with either PBO or TPP changed the acute toxicity level and LE sensitivity compared with those exposed to FNT alone. The acute toxicity level and susceptibility of LE to FNT was found to be inversely related. With additional studies P. parva and G. holbrooki have the potential to be used as bioindicator, and LE as biochemical biomarker of environmental pollution by FNT and other related compounds.
... Oxidative derivation of CPF to 3,5,6-trichloro-2-pyridinol (TCP) and diethylthiophosphate represents a competing detoxification pathway which is likewise mediated by hepatic CYP [11]. A balance between the extent of metabolic activation and detoxification determines individual susceptibility to the toxicity of this compound and is most likely associated with gender and age-dependent sensitivity [11,12]. 0048 Zinc (Zn) is a trace element essential for cell proliferation and differentiation. ...
... This greater capacity for oxidative metabolism can cause the male animals of certain rodent strains to be more or less susceptible to toxicity from a chemical depending on whether oxidative metabolism represents a bioactivation or detoxification pathway for the chemical at the dose it is administered [65]. Supporting this point, Chambers et al. [12] reported that the adult male rats have a higher rate of both hepatic activation to CPF-oxon, the metabolite that inhibits cholinesterase, as well as more rapid detoxification. The results showed that zinc administration to CPF-treated rats reduced the neurotoxic action of CPF and protects AChE from the inhibitory effects of CPF to insignificant effect in female rats and significant (p 6 0.05) in male rats. ...
The protective effects of zinc on liver and kidney injury induced by chlorpyrifos (CPF) were investigated in rats. Male and female rats were orally administered CPF at a dose of 6.75 mg kg−1 body weight for 28 consecutive days. An additional CPF group received zinc (227 mg l−1) in drinking water throughout the experimental duration. Two groups more served as controls. Administration of CPF resulted in a significant increase in serum lipid peroxidation (LPO) level, while induced significant decreases in the activities of plasma superoxide dismutase (SOD), glutathione-S-transferase (GST) and serum acetylcholinesterase (AChE) either in male or female rats. Similarly, a significant increase in the levels of various serum marker enzymes [e.g. aminotransferases (AST and ALT), lactate dehydrogenase (LDH) and gamma glutamyl transferase (GGT)] and increase the level of total protein, uric acid and creatinine. In contrast, co-administration of zinc to CPF-treated animals restored most of these biochemical parameters to within normal levels. In case of AChE, supplementation of zinc showed little alteration in the activity of this enzyme especially in male rats treated with CPF. CPF caused histopathological change in liver and kidneys of male and female rats. However, zinc administration to CPF-treated animals resulted in overall improvement in liver and kidneys damage, emphasizing its antioxidant role. In light of the available data, it can deduce that CPF-induced lipid peroxidation, oxidative stress, liver and kidneys damage in male and female rats, and conjunction supplementation of zinc has resulted in pronounced ameliorating effect.
... make the pesticide ineffective against individuals that naturally have high levels of these enzymes [19]. These esterases detoxify organophosphate (OP) and carbamate pesticides and synthetic pyrethroids by two main ways -hydrolysis of the ester bond and binding of the pesticide (OPs) to the active site [20][21][22]. ...
The present work is focus on physical and chemical properties of purified Carboxylesterase using the Seeds of Tamarindus Indica.The esterases are extracted from the germinating tamarind seeds using 50 mM phosphate buffer, pH 7 and purified. The Km with α-naphthyl acetate, β-naphthyl acetate and α-naphthyl butyrate as the substrates is 28.6 μM, 22.2 μM and 26.7 μM respectively. The Vmax for the same substrates is 7.1 x 10-3 µmole/min, 7.41 x 10-3 µmole/min and 8.00 x 10-3 µmole/min respectively. The enzymes optimally active at pH 7.0 and are stable between pH 5.0 to 8.0. The optimum temperature of esterase activity is 40˚C. The molecular weight of 27.5 kD as determined by SDS-PAGE, both in the presence and absence of β-mercaptothanol and is in close agreement with the molecular weight determined by gel-filtration on Sephadex G-100 (26.9 kD).
... The lowtoxic pyrimidinol (M2) was also obtained from diazinon via M1 with oxidative desulfurization, hydrolysis pathways, which is the main degradation product in some soil, 47,48 plant 49 and animal 44 experiments, as well as documented in other pesticides. [50][51][52] For example, chlorpyrifos can form diethyl phosphate, diethyl phosphorothioate and 2,3,5-trichloro-4-pyridinyl alcohol through the above reactions. 53 In this study, M1 is more toxic than diazinon, and there may be four ways to continue to decompose it. ...
The fungal metabolism of diazinon was investigated and the microbial model (Cunninghamella elegans ATCC36112) could effectively degrade the organophosphorus pesticide (diazinon) mediated by cytochrome P450, which was mainly involved in oxidation and hydrolysis of phase I metabolism. Approximately 89% of diazinon was removed within 7 days and was not observed after 13 days with concomitant accumulation of eight metabolites. Structures of the metabolites were fully or tentatively identified with GC-MS and ¹H, ¹³C NMR. The major metabolites of diazinon were diethyl (2-isopropyl-6-methylpyrimidin-4-yl) phosphate (diazoxon) and 2-isopropyl-6-methyl-4-pyrimidinol (pyrimidinol), and formation of minor metabolites was primarily the result of hydroxylation. To determine the responsible enzymes in diazinon metabolism, piperonyl butoxide and methimazole were treated, and the kinetic responses of diazinon and its metabolites by Cunninghamella elegans were measured. Results indirectly demonstrated that cytochrome P450 and flavin monooxygenase were involved in the metabolism of diazinon, but methimazole inhibited the metabolism less effectively. Based on the metabolic profiling, a possible metabolic pathway involved in phase I metabolism of diazinon was proposed, which would contribute to providing insight into understanding the toxicological effects of diazinon and the potential application of fungi on organophosphorus pesticides.
... The widely used OP chlorpyrifos (CPF, O,O-diethyl-O-[3,5,6-trichloro-2-pyridinyl] phosphorothioate) is metabolized in humans to CPF-oxon, an active metabolite that also acts as a potent anti-cholinesterase [13]. Juvenile rats have lower activities of CPF-detoxifying enzymes than adult rats [14] and thus are more vulnerable to CPF toxicity [15]. ...
The widely used organophosphorus pesticide chlorpyrifos (CPF) is often detected in food. CPF inhibits acetylcholinesterase and can modify muscle contractility and respiratory patterns. We studied the effects of chronic exposure to CPF on respiratory parameters and diaphragm contractility in 21- and 60-days old rats. Pregnant rats were exposed to oral CPF (1 or 5 mg/ kg /day: CPF-1 or CPF-5 groups vs vehicle: controls) from gestation onset up to weaning of the pups that were individually gavaged (CPF or vehicle) thereafter. Two developmental time points were studied: weaning (day 21) and adulthood (day 60). Whole-body plethysmography was used to score breathing patterns and apnea index during sleep. Then, diaphragm strips were dissected for the assessment of contractility and acetylcholinesterase activity. Results showed that the sleep apnea index was higher in CPF-exposed rats than in controls. In adult rats, the expiratory time and tidal volume were higher in CPF-exposed animals than in controls. At both ages, the diaphragm’s amplitude of contraction and fatigability index were higher in the CPF-5 group, due to lower acetylcholinesterase activity. We conclude that chronic exposure to CPF is associated with higher sleep apnea index and diaphragm contractility, and modifies respiratory patterns in sleeping juvenile and adult rats.
... Pesticidedetoxifying enzymes such as CarbE can make the pesticide ineffective against animals that naturally have high levels of these enzymes [5]. In particular, CarbE detoxifies many different compounds of pesticides by hydrolysis of an ester bond, or by binding the pesticide to the active site of CarbE [6]; [7][8][9]. Soluble glutathione transferases (GST) protect the body against electrophilic toxicants by catalyzing their conjugation with glutathione (GSH) (Hinson and [10]. GST plays a crucial role in the maintenance of the intracellular redox state and thus protects the cell against oxidative injuries. ...
Collembolans are small animals live in dead vegetation and consumers of dead plant material. Carboxylesterases (CarbEs) and glutathione transferase (GST) play a key role in the detoxification of many agrochemicals. To gain knowledge on the role of CarbEs and GST activities in the natural tolerance in collembolan insects, we performed enzyme kinetic analyses to determine whether collembolans are able to generate these enzymes in their bodies after exposure to chemicals. The aim of this study also was to explore the effects of repetitive applications of a mixture of pesticides upon CbE and GST of springtails in order to increase the understanding on pesticide detoxification enzymes in this organism. Our studies provide a baseline study on the impact of various agrochemicals on the levels of the detoxification enzymes for collembolans. Application of pesticides such as Bordeaux mixture, imidacloprid, methiocarb and glyphosate resulted in significant increases of CarbEs in collembolan insects. Polyacrylamide gel electrophoresis (PAGE) of CarbE levels revealed visible stronger bands in the response of collembolans to all the applied pesticides, compared to control. A significant increase in glutathione S-transferases (GST) activity was recorded only after application of Bordeaux mixture. The data presented in this study illustrate the impact of various pesticides on the levels of relevant detoxification enzymes such as CarbE and GST in collembolan insects.
... Pesticidedetoxifying enzymes such as CarbE can make the pesticide ineffective against animals that naturally have high levels of these enzymes [5]. In particular, CarbE detoxifies many different compounds of pesticides by hydrolysis of an ester bond, or by binding the pesticide to the active site of CarbE [6]; [7][8][9]. Soluble glutathione transferases (GST) protect the body against electrophilic toxicants by catalyzing their conjugation with glutathione (GSH) (Hinson and [10]. GST plays a crucial role in the maintenance of the intracellular redox state and thus protects the cell against oxidative injuries. ...
Collembolans are small animals live in dead vegetation and consumers of dead plant material. Carboxylesterases (CarbEs) and glutathione transferase (GST) play a key role in the detoxification of many agrochemicals. To gain knowledge on the role of CarbEs and GST activities in the natural tolerance in collembolan insects, we performed enzyme kinetic analyses to determine whether collembolans are able to generate these enzymes in their bodies after exposure to chemicals. The aim of this study also was to explore the effects of repetitive applications of a mixture of pesticides upon CbE and GST of springtails in order to increase the understanding on pesticide detoxification enzymes in this organism. Our studies provide a baseline study on the impact of various agrochemicals on the levels of the detoxification enzymes for collembolans. Application of pesticides such as Bordeaux mixture, imidacloprid, methiocarb and glyphosate resulted in significant increases of CarbEs in collembolan insects. Polyacrylamide gel electrophoresis (PAGE) of CarbE levels revealed visible stronger bands in the response of collembolans to all the applied pesticides, compared to control. A significant increase in glutathione S-transferases (GST) activity was recorded only after application of Bordeaux mixture. The data presented in this study illustrate the impact of various pesticides on the levels of relevant detoxification enzymes such as CarbE and GST in collembolan insects.
... Diazinon (DZN) is a thionophosphorus organophosphate (OP) insecticide whose toxicity is mediated through Cytochrome P450 (CYP) metabolism to DZN-oxon, respectively. The parent OPs are weak inhibitors of AChE, however they undergo CYP-mediated oxidative desulfuration to become potent inhibitors of AChE (Sultatos and Murphy, 1983;Amitai et al., 1998;Chambers et al., 1994). Microsomes derived from intestinal enterocytes demonstrated a similar CYP-mediated metabolism profile involving both desulfuration and dearylation reactions, with dearylation to the pyridinol/pyrimidinol also representing the preferred pathway for metabolism of OPs . ...
Acute poisoning with OPs may lead to a range of neurological effects, which cannot be explained by AChE inhibition alone. Several OPs interact directly with cholinergic receptors in mammals, but such data does not exist for invertebrates. The aim of current study was to investigate the direct and indirect effects of diazinon on the contractions of rat ileum and to compare those effects on the nervemuscle preparation of the Ascaris suum. In the presence of increasing concentrations of diazinon (3, 10 and 30 nM), EFS-induced ileal contractions were increased significantly. In the same preparation, diazinon 3 nM, significantly increased contractions induced by EFS, but did not affect the contractions caused by 5MFI. Contrarily, 1 μM of diazinon significantly and reversibly inhibited the EFS-induced ileal contractions. Diazinon exhibited competitive and non-competitive inhibitions of 5MFI induced contractions. The control EC50 of 5MFI was 2.48 μM with Rmax = 1.88 g. In the presence of diazinon, EC50 was 12.45 μM, while Rmax was reduced to 0.43 g. After washing, the EC50 and Rmax values were again closer to the control level (3.80 μM and 1.04 g). Diazinon 1 μM did not inhibit Ascaris suum contractions caused by ACh, but it increased the Rmax. Diazinon in our study exhibits two opposite effects on the motor activity of the ileum. In low nanomolar concentrations the dominat is its effect on AChE and the stimulation of contractions. Furthermore, in concentrations that approach micromolar values diazinon has a direct inhibitory effect on muscarinic receptors. The direct inhibitory effect of diazinon on A. suum contractions was not found.
... Pesticidedetoxifying enzymes such as CarbE can make the pesticide ineffective against animals that naturally have high levels of these enzymes [5]. In particular, CarbE detoxifies many different compounds of pesticides by hydrolysis of an ester bond, or by binding the pesticide to the active site of CarbE [6]; [7][8][9]. Soluble glutathione transferases (GST) protect the body against electrophilic toxicants by catalyzing their conjugation with glutathione (GSH) (Hinson and [10]. GST plays a crucial role in the maintenance of the intracellular redox state and thus protects the cell against oxidative injuries. ...
Collembolans (springtails) are a group of small animals that live in dead vegetation and leaf litter and in the soil pores and cavities to a depth of about 15 cm. They are important consumers of dead plant material, and eat saprophytic microbes. Carboxylesterases (CbEs) and glutathione-S-transferase (GST) play a key role in the detoxification of many agrochemicals. These enzymes are involved in the biochemical mechanisms underlying resistance to pesticides in some pest species. They also provide an efficient protective mechanism against toxicity caused by several agrochemicals in invertebrates. To gain knowledge on the role of CarbEs and GST activities in the natural tolerance in collembolan insects, we performed enzyme kinetic analyses to determine whether these insects are able to generate these enzymes in their bodies after exposure to various chemicals. However, there are no biochemical studies on the impact of pesticides upon these soil organisms. Our studies provide a baseline study on the impact of various agrochemicals on the levels of the detoxification enzymes CarbE and GST for collembolan insects. Application of pesticides such as Bordeaux mixture, imidacloprid, methiocarb and glyphosate resulted in significant increases of CarbEs in collembolan insects. Polyacrylamide gel electrophoresis (PAGE) of CarbE levels revealed visible stronger bands in the response of collembolans to all the applied pesticides, compared to control. A significant increase in glutathione S-transferases (GST) activity was recorded only after application of 17 Bordeaux mixture.
... However, cytochrome-P-450-mediated detoxification of xenobiotics results in enhanced free radical content of cells. Insecticides may be broken down in different ways, which can lead to different products of a higher or lower toxicity than the mother compound (BRATTSTEN et al. 1986, CHAMBERS et al. 1994. Inhibition of cytochrome P-450 protects rat liver cells from death (YAMANO & MORITA 1992). ...
Spodoptera exigua and Tenebrio molitor larvae were exposed to fenitrothion at concentrations that caused the death of 10% and 30% of treated groups during 4 days. The pesticide caused a decrease in the weight of the fat body. The survival curves were different in the two species. Almost all T. molitor larvae died after the third application of the insecticide, while about 3040% of treated S. exigua larvae survived the stress. The activities of superoxide dismutase and catalase changed after the pesticide application, suggesting that exposure to fenitrothion induces oxidative stress.
... Indirect PON1 assay. Assay of PON1 at submicromolar OP concentrations was accomplished by using inhibition of an exogenous source of AChE (bovine brain) to quantify residual OP following incubation of the OP in human serum containing either 2 mM CaCl 2 (to stimulate PON1 activity) or 1 mM EDTA (to inhibit PON1 activity) using some modifications of our method described earlier (Chambers et al., 1994). OPs were used at concentrations which would yield 80%-90% inhibition of the AChE following incubation in buffer without serum. ...
Paraoxonase 1 (PON1) is a calcium-dependent hydrolase associated with serum high density lipoprotein particles. PON1 hydrolyzes some organophosphates, including some nerve agents, through nucleophilic attack of hydroxide ion (from water) in the active site. Most organophosphates are hydrolyzed inefficiently. This project seeks to identify nucleophiles that can enhance PON1-mediated organophosphate degradation. A series of novel nucleophiles, substituted phenoxyalkyl pyridinium oximes, has been synthesized which enhance the degradation of surrogates of sarin (nitrophenyl isopropyl methylphosphonate; NIMP) and VX (nitrophenyl ethyl methylphosphonate; NEMP). Two types of in vitro assays have been conducted, a direct assay using millimolar concentrations of substrate with direct spectrophotometric quantitation of a hydrolysis product (4-nitrophenol) and an indirect assay using sub-micromolar concentrations of substrate with quantitation by the level of inhibition of an exogenous source of acetylcholinesterase from non-hydrolyzed substrate. Neither NIMP nor NEMP is hydrolyzed effectively by PON1 if one of these novel oximes is absent. However in the presence of eight novel oximes, PON1-mediated degradation of both surrogates occurs. Computational modeling has created a model of PON1 embedded in phospholipid and has indicated general agreement of the binding enthalpies with the relative efficacy as PON1 enhancers. PON1 enhancement of degradation of organophosphates could be a unique and unprecedented mechanism of antidotal action.
... Experiments with mosquitofish (Gambusia affinis), which included methyl parathion (a weak toxicant in fish), along with parathion and chlorpyrifos, also showed correspondence among the potencies of the three oxons for brain AChE inhibition and the toxicity level of the parent insecticide, but no correspondence was seen in the metabolism of the insecticides (Boone and Chambers 1997). We have concluded previously that the acute toxicity levels of OP insecticides in mammals, in contrast to fish, are determined more by differences in metabolic efficiencies than by differences in target enzyme sensitivity (Chambers et al. 1994;Ma and Chambers 1995;Chambers and Carr 1995). ...
Biomarkers are measurable biological parameters that change in response to xenobiotic exposure and other environmental or physiological stressors, and can be indices of toxicant exposure or effects. If the biomarkers are sufficiently specific and well characterized, they can have great utility in the risk assessment process by providing an indication of the degree of exposure of humans or animals in natural populations to a specific xenobiotic or class of xenobiotics. Most biomarkers are effective as indices of exposure, but adequate information is rarely available on the appropriate dose-response curves to have well-described biomarkers of effect that can be widely applicable to additional populations. Specific examples of acetylcholinest-erase inhibition following exposure to organophosphorus insecticides are cited from experiments in both mammals (rats) and fish. These experiments have indicated that the degree of inhibition can be readily influenced by endogenous (e.g., age) and exogenous (e.g., chemical exposures) factors, and that the degree of inhibition is not readily correlated with toxicological effects. Caution is urged, therefore, in an attempt to utilize biomarkers in the risk assessment process until more complete documentation is available on the specificity, sensitivity, and time course of changes, and on the impact of multiple exposures or the time of exposures.
... In the case of diazoxon, the catalytic efficiency is still high (albeit lower than with chlorpyrifos oxon), and no alloform-specific differences are evident. With paraoxon, the PON1 R192 alloform is much more efficient than the PON1 Q192 alloform; however, its overall catalytic efficiency is too low to protect against exposure, confirming the hypothesis (Chambers et al., 1994; Pond et al., 1995) that PON1 may not degrade paraoxon efficiently in vivo. Further support for these conclusions has derived from studies in PON1 transgenic mice. ...
Paraoxonase (PON1) is an A-esterase capable of hydrolyzing the active metabolites (oxons) of a number of organophosphorus (OP) insecticides such as parathion, diazinon and chlorpyrifos. PON1 activity is highest in liver and in plasma. Human PON1 displays two polymorphisms in the coding region (Q192R and L55M) and several polymorphisms in the promoter and the 3'-UTR regions. The Q192R polymorphism imparts differential catalytic activity toward some OP substrates, while the polymorphism at position -108 (C/T) is the major contributor of differences in the levels of PON1 expression. Both contribute to determining an individual's PON1 "status". Animal studies have shown that PON1 is an important determinant of OP toxicity. Administration of exogenous PON1 to rats or mice protects them from the toxicity of specific OPs. PON1 knockout mice display a high sensitivity to the toxicity of diazoxon and chlorpyrifos oxon, but not of paraoxon. In vitro catalytic efficiencies of purified PON(192) alloforms for hydrolysis of specific oxon substrates accurately predict the degree of in vivo protection afforded by each isoform. Evidence is slowly emerging that a low PON1 status may increase susceptibility to OP toxicity in humans. Low PON1 activity may also contribute to the developmental toxicity and neurotoxicity of OPs, as shown by animal and human studies.
... Parathion is metabolized into paraoxon and 4-nitrophenol by desulfuration and dearylation (Fig. 1), respectively. 4-Nitrophenol formation is considered as the inactivation pathway, whereas paraoxon formation is considered as an activation pathway (Benke GM 1975;Chakravarty 1986, 1988;Butler andMurray 1993, 1997;Chambers and Forsyth 1989;Chambers et al. 1994;Chaturvedi et al. 1991;Contreras et al. 1999;Halpert et al. 1980;Halpert and Neal 1981a, b;Hou et al. 1996;Kulkarni and Hodgson 1982;Kuo and Perera 2000;Lapadula et al. 1984;Levi and Hodgson 1985;Martinez-Zedillo et al. 1979;Morgan et al. 1994;Butler 1994, 1995;Mutch et al. 1999;Mutch et al. 2003;Nadin and Murray 1999;Purshottam and Srivastava 1987;Ramos and Sultatos 1998;Rowland et al. 1991;Soranno and Sultatos 1992;Sultatos 1986;Sultatos et al. 1984;Sultatos and Gagliardi 1990;Sultatos and Minor 1986;Sultatos and Murphy 1983;Tang and Chambers 1999;Vargas Loza et al. 1997;Venera et al. 1978;Vitarius et al. 1995;Wallace and Dargan 1987;Watson et al. 1994;Zhang and Sultatos 1991;Zhu and Liu 1994). The primary metabolizing organ is the liver by the enzyme cytochrome P450 3A4. ...
Introduction Physiologically Based Pharmacokinetic Modeling Pharmacodynamic Modeling Future Directions References
... Remarkably these traits appeared to be significant in all different combinations and in this case likely interactions with more than four traits might be applicable and appropriate. Elimination abilities have yet not been subject to many comparative studies and are mostly related to physiological traits investigating detoxification, hence relating enzyme activities with effect responses (Chambers et al. 1994; Chambers and Carr 1995; Printes and Callaghan 2004; Domingues et al. 2010). As no detoxification traits were included in this study (see above), it is difficult to compare the results found to existing knowledge in literature. ...
Ecological risk assessment (ERA) has followed a taxonomy-based approach, making the assumption that related species will show similar sensitivity to toxicants, and using safety factors or species sensitivity distributions to extrapolate from tested to untested species. In ecology it has become apparent that taxonomic approaches may have limitations for the description and understanding of species assemblages in nature. Therefore it has been proposed that the inclusion of species traits in ERA could provide a useful and alternative description of the systems under investigation. At the same time, there is a growing recognition that the use of mechanistic approaches in ERA, including conceptual and quantitative models, may improve predictive and extrapolative power. Purposefully linking traits with mechanistic effect models could add value to taxonomy-based ERA by improving our understanding of how structural and functional system facets may facilitate inter-species extrapolation. Here, we explore whether and in what ways traits can be linked purposefully to mechanistic effect models to predict intrinsic sensitivity using available data on the acute sensitivity and toxicokinetics of a range of freshwater arthropods exposed to chlorpyrifos. The results of a quantitative linking of seven different endpoints and twelve traits demonstrate that while quantitative links between traits and/or trait combinations and process based (toxicokinetic) model parameters can be established, the use of simple traits to predict classical sensitivity endpoints yields little insight. Remarkably, neither of the standard sensitivity values, i.e. the LC50 or EC50, showed a strong correlation with traits. Future research in this area should include a quantitative linking of toxicodynamic parameter estimations and physiological traits, and requires further consideration of how mechanistic trait-process/parameter links can be used for prediction of intrinsic sensitivity across species for different substances in ERA.
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... Therefore the observed site differences in ROO or TOT TOSC in A. purpurea chicks agrees with previous studies and could be related to exposure to contaminants such as Hg, PCBs or DDTs. Plasmatic B esterases like those in liver are known to act as alternative target sites for anticholinergic compounds, thus protecting brain and muscle cholinesterases from inhibitory substances (Chambers et al., 1994). Activity levels of B esterases were similar across sites in both species, thus indicating low or no exposure to anticholinergic compounds. ...
Blood biomarkers and levels of major pollutants in eggs and feathers were used to determine pollution effects in nestlings of the Purple Heron Ardea purpurea and the Little Egret Egretta garzetta, sampled on three Ebro River (NE Spain) areas: a reference site, a site affected by the effluents of a chlor-alkali industry and the river Delta. The two impacted heron populations showed mutually different pollutant and response patterns, suggesting different sources of contamination. In the population nesting near the chlor-alkali plant, elevated levels of hexachlorobenzene (HCB) and polychlorobiphenyls (PCBs) in eggs, and mercury in feathers in A. purpurea chicks were related with reduced blood antioxidant defenses and increased levels of micronuclei. In Ebro Delta, high levels of plasmatic lactate dehydrogenase in A. purpurea chicks and high frequency of micronuclei in blood of both species were tentatively associated with intensive agricultural activities taking place in the area. These results provide the first evidence of a biological response in heron chicks to the release of pollutants at a chlor-alkali plant.
Organophosphates (OP) inhibit serine hydrolases by phosphorylating serine residue. Exposure to OPs always involves acetylcholine esterase (AChE) inhibition, which is responsible for the degradation of acetylcholine (ACh), controlling the levels of ACh at the nerve endings. Inactivation/inhibition of AChE causes excessive accumulation of ACh at the neuromuscular junctions and synapses activating both sympathetic and parasympathetic processes causing both muscarinic and nicotinic toxicity. The muscarinic symptoms include salivation, diaphoresis, abdominal cramps, diarrhea, vomiting, miosis, bronchorrhea, bradycardia, coma, and seizure. Nicotinic symptoms are fasciculations, flaccid paralysis, and tachycardia. The most common cause of death is respiratory failure with refractory hypotension. Recovery from OP toxicity is linked to the restoration of OP‐inhibited AChE, which can be revived by oximes or regeneration of the enzyme, certain OPs bind irreversibly with AChE render it permanently inactive. The secondary OP targets in humans are butyrylcholinesterase (BChE), a sink for OP, and neuropathy target esterase, inhibition of which causes delayed neuropathy. The OPs are not only used for the management of pests in agriculture but also used in the treatment of human diseases, such as glaucoma with echothiophate to increase drainage of intraocular fluid reducing ocular pressure, schistosomiasis, and Alzheimer's with metrifonate. Metrifonate inhibits AChE in schistosomes, which is abundant in the muscles of the parasite. Metrifonate enhances central nervous system (CNS) cholinergic neurotransmission improving cognitive function in Alzheimer's patients. This chapter describes the AChE inhibition and toxicity of each OP and regeneration of AChE by oximes along with regulations based on the available data for individual OPs.
Organophosphate (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxication can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON1) are considered factors underlying age-related sensitivity differences. We used an in vitro system to measure detoxication of AChE-inhibiting pesticides mediated via these esterases. Recombinant human AChE was used as a bioassay of inhibitor concentration following incubation with detoxifying tissue: liver plus Ca(+2) (to stimulate PON1s, measuring activity of both esterases) or EGTA (to inhibit PON1s, thereby measuring CaE activity). AChE inhibitory concentrations of aldicarb, chlorpyrifos oxon, malaoxon, methamidophos, oxamyl, paraoxon, and methylparaoxon were incubated with liver homogenates from adult male rat or one of 20 commercially provided human (11-83 years of age) liver samples. Detoxication was defined as the difference in inhibition produced by the pesticide alone and inhibition measured in combination with liver plus Ca(+2) or liver plus EGTA. Generally, rat liver produced more detoxication than did the human samples. There were large detoxication differences across human samples for some pesticides (especially malaoxon, chlorpyrifos oxon) but not for others (e.g., aldicarb, methamidophos); for the most part these differences did not correlate with age or sex. Chlorpyrifos oxon was fully detoxified only in the presence of Ca(+2) in both rat and human livers. Detoxication of paraoxon and methylparaoxon in rat liver was greater with Ca(+2), but humans showed less differentiation than rats between Ca(+2) and EGTA conditions. This suggests the importance of PON1 detoxication for these three OPs in the rat, but mostly only for chlorpyrifos oxon in human samples. Malaoxon was detoxified similarly with Ca(+2) or EGTA, and the differences across humans correlated with metabolism of p-nitrophenyl acetate, a substrate for CaEs. This suggests the importance of CaEs in malaoxon detoxication. Understanding these individual differences in detoxication can inform human variability in pesticide sensitivity.
Chlorpromazine (CPZ), an antipsychotic drug, was found to inhibit intracellular carboxylesterases (CarbEs). As intracellular target carboxylesterases we used alpha-naphthyl acetate esterase (alpha-NA), naphthol AS-D chloroacetate esterase (AS-D) and alpha-naphthyl butyrate esterase (alpha-NB) in mouse polymorphonuclear neutrophils (PMN), hepatocytes (HC) and neuronal brain cells (NC). The impact of CPZ on the cells ranged from no effect to death, with intermediary effects of decreased CarbEs activities without either morphological changes or structural changes. The results of our study indicate that intracellular CarbEs activity inhibition by CPZ was dose-dependent, though the drug concentration required to bring about 50% inhibition of the initial activity (ID-50) varied between the mouse cell types, under the same experimental conditions. CarbEs activity was decreased or completely inhibited at CPZ concentrations ranging from 0.5 to 5.0 mg/ml (1.4 to 14.08 mmol/l). The impact maximum concentration of CPZ 5 mg/ml (14.08 mmol/l) on mouse brain cells resulted in 46.58% inhibition for AS-D, 54.26% for alpha -NA and 99.52% for alpha -NB. Our studies established a clear relationship between the increasing concentrations of CPZ and the extent of inhibition of the intracellular esterases of mice. Correlation of the inhibitory effects in all the cell types was demonstrated. The polymorphonuclear neutrophils - leukocytes were the most sensitive (ID-50 = 0.42 mg CPZ/ml) and the hepatocytes most resistant to the CPZ effect (ID-50 = 2.45 mg CPZ/ml). Since leukocytes are human cells much more readily available than hepatocytes or neuronal cells, we presume that CarbEs in peripheral blood leukocytes could be used as markers for the indication of intracellular biochemical damage of hepatocytes and neuronal brain cells by CPZ.
Chlorpromazine, an antipsychotic drug, is found to inhibit intracellular esterase. As intracellular target esterases we used alpha-naphthyl acetate esterase, naphthol AS-D chloroacetate esterase and alpha-naphthyl butyrate esterase in polymorphonuclear neutrophyls, hepatocytes and neuronal cells of mouse. In vitro study showed that chlorpromazine affects esterases of all these cell types whereby the degree of the enzyme inhibition was dependent on the incubation time and drug concentration. The correlation of this inhibitory effect in all the cell types, has been demonstrated whereby the polymorphonuclear neutrophyls were proved to be the most sensitive to chlorpromazine effect.
The acute toxicity of organophosphorus (OP) nerve agents and pesticides is due to inhibition of acetylcholinesterase (AChE) activity in cholinergic nerve synapses. Delayed neuropathy involves OP binding to neuropathy target esterase (NTE) in neurons. AChE and NTE are serine hydrolases. In vitro studies have demonstrated that OP toxicants bind not only to serine hydrolases but also to proteins that have no active site serine, for example, Tyr 411 of human albumin and Lys 296 of mouse transferrin. Mice treated with low doses of chlorpyrifos have OP-modified tubulin in brain. Mass spectrometry has identified OP-modified albumin in the blood of humans self-poisoned by chlorpyrifos and dichlorvos. Albumin is weakly reactive with OP, but the presence of OP-modified albumin in humans suggests the existence of additional noncholinesterase OP targets. In conclusion, long-term adverse effects from OP exposure may involve OP modification of proteins that have no active site serine.
Parathion and chlorpyrifos are phosphorothionate insecticides, and parathion is about an order of magnitude more toxic than chlorpyrifos. Our laboratories have investigated several aspects of the biochemical reactivity and the metabolism of these two insecticides in rats to identify factors that influence the acute toxicity level. Both insecticides are bioactivated by cytochromes P450 to potent oxon metabolites by desulfuration, with bioactivation of parathion more efficient than that of chlorpyrifos. P450-mediated detoxication, dearylation, can also occur, and is more effective against chlorpyrifos than parathion. The oxons can persistently inhibit serine esterases, including nervous system acetylcholinesterase (the target enzyme for acute toxicity) and protective B-esterases such as carboxylesterases; chlorpyrifos-oxon is a more potent inhibitor of both acetylcholinesterase and carboxylesterases than paraoxon. The oxons can be hydrolyzed by paraoxonase (PON) which is much more efficient towards chlorpyrifos-oxon than paraoxon. Chlorpyrifos, because of its high lipophilicity, results in more persistent inhibition of serine esterases than does parathion. The lower toxicity of chlorpyrifos than parathion seems to be the result largely of less effective bioactivation and more effective detoxication by P450, carboxylesterases and paraoxonase, despite the greater affinity of chlorpyrifos-oxon than paraoxon toward acetylcholinesterase. These factors can be useful in PBPK modeling.
Polymorphisms in the paraoxonase (PON1) gene influence both the quality and the quantity of PON1. Evidence provided by animal studies indicates that PON1 plays a relevant role in the metabolism of certain organophosphates (OPs), and modulates their toxicity and developmental neurotoxicity. Careful in vitro studies carried out under physiological conditions, together with in vivo studies in various lines of PON1 transgenic mice, have been shown to be extremely useful in dissecting the functional significance of PON1. The influence of PON1 status on inferring susceptibility or protection toward OPs needs to be evaluated for each individual compound. It is somewhat ironic that the toxicity of paraoxon, the OP after which PON1 was named, is not significantly influenced by PON1 status. Although there is strong evidence to indicate that PON1 levels and, in some cases, the Q192R polymorphism determine the efficiency with which an individual will detoxify a specific OP; however, further proof in human populations is still needed. In particular, studies are needed in which PON1 status is correlated with the degree of exposure, and with signs and symptoms of toxicity. Animal studies have also pointed out the potential therapeutic use of PON1 in treating individuals with exposure to OP insecticides and/or nerve agents. The recent expression of active human PON1 in Escherichia coli and the elucidation of PON1 structure provide the necessary breakthroughs for producing, recombinant variants that have catalytic efficiency sufficient for therapeutic applications.
The chapter provides an overview of the characteristics of enzymes-catalyzing pesticide metabolism, and common metabolic pathways of organophosphorus (OP) and carbamate (CM) pesticides. Also, it presents a recent trend of using human tissue for the in vitro study of pesticide metabolism, which may provide more relevant data to human health. The study of xenobiotic metabolism focuses on the recognition of enzymes involved and identification of metabolites generated as well as the understanding of the effects of xenobiotics on the metabolic enzymes. Several families of metabolic enzymes, often with wide arrays of substrate specificity, are involved in OP and CM pesticide metabolism. These metabolic enzymes are often divided into two distinct groups, referred to as phase I and phase II enzymes. Phase I enzymes introduce a polar reactive group onto the molecule, making it more water soluble while also increasing the possibility for further metabolism by phase II enzymes. More often than not, these metabolic processes are detoxication reactions. However, there are some cases in which metabolism through either phase I or phase II can make the chemical more reactive than the parent compound. This is particularly true in the case of OP compounds, in which the conversion of a P=S moiety to a P=O group can result in increasing toxicity substantially. Usually, however, metabolic events that increase the water solubility of a chemical cause significant reductions in its biological half-life by making it more readily excreted.
PON1 is a member of a family of proteins that also includes PON2 and PON3, the genes of which are clustered in tandem on the long arms of human chromosome 7 (q21.22). Paraoxonase (PON1) is synthesized primarily in the liver and a portion is secreted into the plasma, where it is associated with high-density lipoproteins (HDL). PON1, which received its name from its ability to hydrolyze paraoxon, its first and the most studied organophosphate (OP) substrate, hydrolyzes the active metabolites of several other OP insecticides, as well as nerve agents such as sarin, soman, and VX. The crystal structure for a recombinant PON1 indicates that it is a six-bladed b-propeller, with two calcium ions in the central tunnel, one of which is essential for enzyme activity and the other for enzyme stability. Evidence that PON1 plays a role in modulating the toxicity of OPs in vivo has emerged slowly over the last 30 years. Initial indirect evidence was provided by comparison across animal species, which differ in the levels of their plasma PON1 activity. Birds that have very low PON1 activity display a much higher sensitivity, compared to rats, to the acute toxicity of some OPs. Current therapy for OP poisoning relies on the use of atropine, a cholinergic muscarinic antagonist, and on oximes, such as pralidoxime (2-PAM), to reactivate phosphorylated. Anticonvulsant drugs such as diazepam may also be utilized to control OP-induced convulsions.
The chapter discusses how various species differ in response to anticholinesterases (anti-ChEs), and proposes potential causes, focusing on pharmacokinetics and pharmacodynamics (i.e., how the body handles the compound and the mechanism of action of the compound). Cholinesterase (ChE) inhibition by organophosphorus compounds (OPs), and to a lesser extent carbamates (Cms), has been studied extensively as the primary mechanism of toxicity for these broad-spectrum insecticides. Although several factors must be considered in toxicology testing of anti-ChEs, including the specific compound in question, species, and age, as well as level and duration of exposure, the chapter focuses on interspecies variability. Various species respond differently to anti-ChEs. Response, recovery, and reversal depend on both the species affected and the compound. Abundant studies exist on various species, from the intended target group (insects) to animals vulnerable to unsolicited effects, including fish, amphibians, birds, and mammals. These interspecies comparisons seek to improve species selectivity, and to extrapolate toxicity testing between unrelated species for regulatory purposes. First, to improve selectivity for target species, it would be helpful to exploit the differences between species. Second, similarities among unrelated species provide insight into conserved mechanisms of action and toxicity. The extrapolation of toxic effects from animal testing to humans may impact human health and safety by providing a basis for setting reference dose levels. At present, uncertainty factors are applied to account for interspecies variability from experimental animals to humans, as well as intraspecies variability to account for the sensitive individuals within a species.
This chapter discusses the metabolism of organophosphorus insecticides. The potency of the organophosphorus insecticides or their active metabolites as inhibitors of target brain acetylcholinesterase does not correspond to the acute toxicity levels, indicating that metabolism and disposition are of great significance in determining the overall acute toxicity level of these insecticides. These insecticides display substantial chemical diversity, including a variety of atoms in addition to the carbon and phosphorus required by the compounds being "organophosphorus" compounds, such as sulfur, nitrogen, and oxygen. Therefore, the organophosphorus insecticides are subject to many metabolic pathways mediated by several of the groups of xenobiotic metabolizing enzymes. The organophosphorus insecticides or their metabolites are subject to phase 1 reactions (oxidations, reductions, hydrolyses) and phase 2 reactions (conjugations). Because of their metabolic and chemical lability, they do not readily remain intact either in the environment or in the organism. Their environmental lability was one of the factors that allowed them to replace the highly stable organochlorine insecticides as the dominant class of insecticides. Some of the organophosphorus insecticides are active anticholinesterases, and any metabolism is therefore a detoxication. Many of the insecticides, however, are not active anticholinesterases in their parent form and require bioactivation in order to be effective anticholinesterases.
Early studies on different human populations showed that the hydrolytic activity of serum paraoxonase (PON1) was polymorphically distributed, raising the hypothesis that individuals with low PON1 activity may be at higher risk from toxic effects due to exposure to organophosphorus (OP) insecticides. Evidence that this may be the case has been obtained from a number of animal studies. First, animal species with low serum PON1 activity (e.g. birds) are especially sensitive to OP toxicity. Among mammals, the rabbits, which have a high serum PON1 activity, are more resistant to OP toxicity than rats. Second, injection of purified PON1 into rats and mice protects them against the acute toxic effects of various OPs. Third, PON1 knockout mice are more sensitive than wild type animals to the toxicity of the oxygen analogs of OPs, such as chlorpyrifos oxon and diazoxon, and resistance can be restored by injecting purified PON1. Surprisingly, PON1 null mice do not show an increased sensitivity to the toxicity of paraoxon, the substrate after which PON1 was named. In vitro studies on catalytic efficiency of PON1, assayed at physiological conditions, indicated that PON1 may be relevant for the detoxication, and hence influence the toxicity, of some OPs (e.g. chlorpyrifos oxon, diazoxon), but not of paraoxon. The increased sensitivity of young animals, and possibly children, to the toxicity of OPs, may be explained in some cases (e.g. chlorpyrifos oxon) by low PON1 activity.
Most organophosphorus compounds today fall into two general groups: the so‐called nerve agents, that are very acutely toxic and organophosphorus pesticides that are less toxic. Nerve agents were generally the first toxic organophosphorus developed, and were the original basis for organophosphorus pesticides. Interest about nerve agents has increased lately given concerns about their potential use in terrorist acts. Organophosphate nerve agents and pesticides are a highly diverse group of chemicals. They are all characterized by their ability to inhibit the enzyme acetylcholinesterase (AChE) that deactivates the neurotransmitter acetylcholine (ACh).
At present, the widest use of organophosphorus compounds is as pesticides, although they have also been used as therapeutic agents, gasoline additives, hydraulic fluids, cotton defoliants, fire retardants, plastic components, growth regulators, and industrial intermediates to a much smaller extent.
Compounds in this class are numerous and have been categorized in many ways according to the nature of the substituents. Gallo and Lawryk (1991) [2], for example, categorized them into four main groups I–IV based on the characteristics of the leaving group (X). Group I compounds, phosphorylcholines, have a leaving group that contains a quaternary nitrogen and are among the most potent organophosphates (e.g., Shradan). Group II compounds, fluorophosphates, have a fluoride leaving group and are also generally highly toxic (e.g., diisopropyl fluorophosphate). Group III compounds have leaving groups that contain cyanide or a halogen other than fluoride and are generally less potent than group I or II (e.g., Parathion). Group IV contains most of the organophosphates used as insecticides today. These compounds have alkoxy, alkylthio, aryloxy, arylthio, or heterocyclic leaving groups and a wide variety of other substituents.
Another classification scheme is based on the nature of the atoms that immediately surround the central phosphorus atom and results in 14 different categories. According to this scheme, phosphates are the prototype for the entire class and are those compounds where all four atoms that surround the phosphorus atom are oxygen (e.g., dichlorvos, mevinphos). Sulfur‐containing organophosphate compounds (phosphorothioates, phosphorothiolates, phosphorodithioates, and phosphorodithiolates) are far more numerous than phosphates and include well‐recognized organophosphate insecticides such as parathion, diazinon, chlorpyrifos, etc. Other groups contain nitrogen (phosphoramides and phosphorodiamides), nitrogen and sulfur (phosphoramidothionates and phosphoramidothiolates), carbon (phosphonates and phosphinates), or carbon and sulfur (phosphonothionates, phosphonothionothiolates, and phosphinothionates).
All aspects of organophosphate chemistry, toxicity, analysis, and exposure potential have been previously and comprehensively reviewed. In addition, information regarding the toxicity of organophosphorus pesticides in particular has expanded greatly in recent years as a result of toxicity data supplied by registrants to the U.S. EPA's Office of Pesticides to support reregistration. These data have been made publicly available by the U.S. EPA on its Internet Web site ( www.epa.gov/pesticides ). The following discussion draws heavily from recent reviews and also includes summaries of relevant toxicity data submitted to, and made available by, the U.S. EPA.
The mortality rate of suicidal parathion poisoning is particularly high, the onset of fulminant cholinergic signs, and the patients frequently present to the emergency physician with life-threatening symptoms. Despite this uniformity, subsequent clinical course differs significantly among patients, mostly not as a result of different delays in treatment or insufficiency of primary care. Probably, the differences depend on the amount of poison absorbed and/or the disposition of the active poison, paraoxon. We followed the toxicokinetics of parathion and tried to quantify the actual poison load. To this end, we monitored parathion-intoxicated patients (patients requiring artificial ventilation) for plasma levels of parathion and paraoxon along with the activity of erythrocyte acetylcholinesterase and its reactivatability. Plasma obidoxime concentrations were followed as well as the cumulative urinary para-nitrophenol conjugate excretion as a measure of total poison load. All patients received a standard obidoxime scheme of a 250mg bolus dose intravenously, followed by continuous infusion with 750mg per 24 hours as long as reactivation could be expected (usually 1 week). All other treatment was instituted as judged by the physician. It was recommended to use atropine at low doses to achieve dry mucous membranes, no bronchoconstriction and no bradycardia. Usually 1–2 mg/h were sufficient. Seven selected cases are presented exemplifying toxicokinetic peculiarities. All patients were severely intoxicated, while the amount of parathion absorbed varied widely (between 0.12 and 4.4g; lethal dose 0.02–0.1g) and was generally much lower than anticipated from the reports of relatives. It remains open whether the discrepancies between reports and findings were due to exaggeration or to effective decontamination (including spontaneous vomiting, gastric lavage and activated charcoal). Absorption of parathion from the gastrointestinal tract was sometimes retarded, up to 5 days, resulting in fluctuating plasma profiles. The volume of distribution at steady-state (Vdss) of parathion was around 20 L/kg. Post-mortem analysis in one patient revealed a 66-fold higher parathion concentration in fat tissue compared with plasma, 16 days after ingestion. Biotransformation of parathion varied widely and was severely retarded in one patient receiving fluconazole during worsening of renal function, while phenobarbital (phenobarbitone) sedation (two cases) had apparently no effect. The proportion of plasma parathion to paraoxon varied from 0.3–30, pointing also to varying paraoxon elimination, as illustrated by one case with particularly low paraoxonase-1 activity. Obidoxime was effective at paraoxon concentrations below 0.5μM, provided aging was not too advanced. This concentration correlated poorly with the parathion concentration or the poison load. The data are discussed in light of the pertinent literature.
The legacy organochlorine insecticide, dieldrin, is still found in soil and accumulation in individuals is possible. Paraoxonase 1 hydrolyzes the oxon metabolites of organophosphorus insecticides, as well as other substrates. Putative binding sites for pregnane X receptor (PXR) exist in the paraoxonase promoter, and studies have indicated that dieldrin can activate PXR-regulated gene expression. We examined rat paraoxonase promoter activity in the presence of dieldrin alone or combined with nuclear receptors (NRs). In vitro dieldrin concentrations from 10 to 100 µM significantly increased (p < 0.05) promoter activity in the presence of Pxr or Rxrα alone and when Pxr plus Rxrα were on the same vector, indicating that dieldrin can increase paraoxonase promoter activity in the presence of NRs. To our knowledge, this is the first report of dieldrin increasing paraoxonase promoter activity. Since many organochlorine insecticides are in the same chemical class as dieldrin, these results could be typical of other bioaccumulative persistent pollutants.
A tiered risk assessment was conducted for the use of granular and liquid formulations of chlorpyrifos in corn agroecosystems in the U.S. The initial screening Tier I assessment suggested that under high-exposure scenarios the granular and some spray formulations present potential hazards to birds. Higher tiered probabilistic risk assessments were conducted separately for the granular and liquid formulations. The probabilistic assessment indicated that risk to birds from exposure to granular formulation is small and that this route of exposure would not be a significant source of mortality. Similarly, the assessment of potential exposure of birds to food items contaminated with chlorpyrifos showed that the risk from exposure via food was small, even if it was assumed that birds feed only on the treated fields. Although they have potentially greater sensitivity to chlorpyrifos, effects in nestling birds consuming food items from fields treated with granular chlorpyrifos were negligible. However, risks to young birds may be greater where the major source of food is from fields treated with liquid formulations of chlorpyrifos. A review of field studies showed that wildlife mortality incidents associated with use of either granular or liquid formulations of chlorpyrifos are not widely apparent in agroecosystems. Based on the multiple lines of evidence, we conclude that the presumption that chlorpyrifos use in corn agroecosystems will result in extensive mortality of terrestrial wildlife, particularly birds and mammals, is not supported by the scientific evidence.
Paraoxonase (PON1) is an HDL-associated enzyme capable of hydrolyzing multiple substrates, including several organophosphorous insecticides and nerve agents, oxidized lipids, and a number of drugs or pro-drugs. Several polymorphisms in the paraoxonase (PON1) gene have been described, which have been shown to affect either the catalytic efficiency of hydrolysis or the expression level of PON1. This review discusses the relevance of these polymorphisms for modulating sensitivity to organophosphorous compounds. Animal studies characterizing the PON1 polymorphisms have demonstrated the relevance of PON1 in modulating OP toxicity and have indicated the importance of an individual's PON1 status (i.e., genotype and phenotype taken together) rather than genotyping alone. Nevertheless, direct confirmation in humans of the relevance of PON1 status in conferring susceptibility to OP toxicity is still elusive. Recent studies examining the involvement of PON1 status in determining OP susceptibility of Gulf War veterans, sheep dippers, and individuals poisoned with chemical warfare agents represent a step in the right direction, but more studies are needed, with better documentation of both the level of exposure and the consequences of exposure.
Biotransformation of terbufos in rat liver revealed four metabolites in the effluent prepared with a C 18 cartridge after the rat liver was perfused for one hour in situ. Analysing the spectrogram of GC-IR and GC-MS, metabolite IV appeared to be an oxidative desulfuration product of terbufos with the formula C 9H 21O 3PS 2, the recovery of which in the effluent was 2.13%; metabolite I appeared to be an hydrolysate of metabolite IV with the formula C 5H 13O 3PS, the recovery of which was 0.13%; metabolite II appeared to be an hydrolysate of terbufos with the formula C 5H 13O 2PS 2, the recovery of which was 2.65%; metabolite III appeared to be a methylate of metabolite II, with the formula C 6H 15O 2PS 2, the recovery of which was 1.42%. Relatively the recovery of terbufos was 40.8%. These results were in accord with the regular metabolic pattern in vivo of phosphorothloates with a thioether group.
Organophosphate pesticides are a highly diverse group of chemicals to which workers may be exposed during manufacture and formulation and during or after application for their intended uses. They are all characterized by their ability to inhibit the enzyme acetylcholinesterase (AChE) that deactivates the neurotransmitter acetylcholine (ACh).
Compounds in this class are numerous and have been categorized in many ways according to the nature of the substituents. Gallo and Lawryk, for example, categorized them into four main categories (Groups I‐IV) based on the characteristics of the leaving group (X). Group I compounds, phosphorylcholines, have a leaving group that contains a quaternary nitrogen and are among the most potent organophosphates (e.g., Shradan). Group II compounds, fluorophosphates, have a fluoride leaving group and are also generally highly toxic (e.g., diisopropyl fluorophosphate). Group III compounds have leaving groups that contain cyanide or a halogen other than fluoride and are generally less potent than Groups I or II (e.g., Parathion). Group IV contains most of the organophosphates used as insecticides today. These compounds have alkoxy, alkylthio, aryloxy, arylthio or heterocyclic leaving groups and a wide variety of other substituents.
Another classification scheme is based on the nature of the atoms that immediately surround the central phosphorus atom and results in 14 different categories. According to this scheme, phosphates are the prototype for the entire class and are those compounds where all four atoms that surround the phosphorus atom are oxygen (e.g., dichlorvos, mevinphos). Sulfur‐containing organophosphate compounds (phosphorothioates; phosphorothiolates; phosphorodithioates; phosphorodithiolates) are far more numerous than phosphates and include well recognized organophosphate insecticides such as parathion, diazinon, chlorpyrifos, etc. Other groups contain nitrogen (phosphoramides and phosphorodiamides), nitrogen and sulfur (phosphoramidothionates and phosphoramidothiolates), carbon (phosphonates and phosphinates), or carbon and sulfur (phosphonothionates, phosphonothionothiolates and phosphinothionates).
All aspects of organophosphate chemistry, toxicity, analysis, and exposure potential have been previously reviewed. Additionally information regarding the toxicity of this class of compounds has expanded greatly in recent years as a result of toxicity data supplied by registrants to the U.S. EPA's Office of Pesticides to support reregistration. These data are being made publically available by the U.S. EPA on their internet web site. The following discussion draws heavily from recent reviews but also includes summaries of relevant toxicity data submitted to the U.S. EPA available when this chapter was completed. Due to space limitations detailed data reviews are included here for only 30 organophosphate pesticides. Information on other pesticides registered or undergoing reregistration in the United States can be readily obtained from the previously mentioned website.
IntroductionPON1 Structure and PolymorphismsPON1 Genotype/Phenotype; Defining PON1 StatusPON1 and OP Toxicity: Animal StudiesPON1 Modulates the Toxicity of a Mixture of OPsPON1 Status and OP Toxicity: Evidence in HumansPON1 and Developmental Toxicity and Human EvidencePON1 as a Catalytic Scavenger for OP PoisoningConclusions
AcknowledgmentsReferences
Paraoxonase (PON1) is a high density lipoprotein-associated enzyme capable of hydrolyzing multiple substrates, including several
organophosphorus (OP) insecticides and nerve agents, oxidized lipids and a number of drugs or pro-drugs. Several polymorphisms
in the PON1 gene have been described, which have been shown to affect either the catalytic efficiency of hydrolysis or the
expression level of the enzyme. Animal studies have shown that PON1 is an important determinant of the toxicity of certain
OPs. Evidence for this was provided by cross-species comparisons, by administration of exogenous PON1 and by experiments in
PON1 knockout and transgenic mice. Low PON1 plays also a role in the higher susceptibility of the young to OP toxicity. Recent
findings also suggest that PON1 may modulate the toxicity resulting from exposure to mixtures of OP compounds
Early research on population distributions of plasma PON1 paraoxonase activity indicated a polymorphic distribution with high,
intermediate and low metabolizers. Cloning and characterization of the cDNA encoding human PON1 and follow-on experiments
demonstrated that the molecular basis of the activity polymorphism (PM) was a Q192R PM with PON1R192 specifying high paraoxonase activity. Further research demonstrated that the PON1192 polymorphism had little effect on the catalytic efficiencies of hydrolysis of phenylacetate and diazoxon (DZO), but did affect
the efficiencies of hydrolysis of chlorpyrifos oxon (CPO), soman and sarin, with PON1R192 having a higher efficiency of CPO hydrolysis and PON1Q192 having higher rates of hydrolysis of soman and sarin. Plots of rates of DZO hydrolysis (at a salt concentration that differentially
inhibited PON1R192) vs. paraoxon hydrolysis clearly separated the three PON1192 phenotypes (QQ, QR, RR) and also showed a wide range of activity among individuals with the same PON1192 genotype. The term PON1 status was introduced to include both PON1192 functional genotype and plasma PON1 level,both important in determining risk for either exposure to specific organophosphorus
compounds (OPs) or disease. Characterization of 5 promoter-region polymorphisms by several groups indicated that an Sp1 binding
site was responsible for significant(~30%) variation in plasma PON1 levels. Re-sequencing of the PON1 genes of 47 individuals
(24 African-American/23 European) revealed an additional 180 polymorphisms in 27 kb of the PON1 genomic DNA including 8 more
5' regulatory region PMs, 1 coding region polymorphism (W194X), 162 additional intronic PMs and 9 additional 3' UTR PMs. The generation of PON1 null mice and “PON1 humanized mice” expressing either tgHuPON1R192 or tgHuPON1Q192 at the same levels on the PON1−/− background allowed for a functional analysis of the Q192R PM under physiological conditions.
Toxicology experiments with the PON1 humanized mice and the PON1 null mice injected with purified human PON1192 alloforms clearly demonstrated that the catalytic efficiency of substrate hydrolysis is important in determining whether
PON1 is able to protect against a given OP exposure. HuPON1R192 protects well against CPO and DZO exposure, but HuPON1Q192 does not protect well against CPO exposure and neither protects
against PO exposure. Studies on PON1 status and carotid artery disease show that low PON1 levels are a risk factor. The effects
of PON1192 alloforms on rates of hydrolysis of quorum sensing factors are not yet known. Taken together, these data along with those
of the leading researchers in the PON1 field indicate that it is important to measure PON1 levels/activities in any epidemiological
study. SNP analysis alone is inadequate for epidemiological studies, due to the wide variability of PON1 levels within the
three PON1192 genotypes Q/Q, Q/R R/R). Even the most comprehensive PON1 SNP analyses are unable to accurately predict PON1 levels. PON1
activity or level accurately predicts CHD risk, while genotype does not
Freshwater fish, topmouth gudgeon (Pseudorasobora parva), were pretreated with piperronyl butoxide (PBO) or triphenyl phosphate (TPP) and then exposed to different concentrations
of fenitrothion (FNT) in a static system. Evaluation of brain acetylcholinesterase (AchE) activity after 24, 48 and 96 h pesticide
exposure indicated that AchE activity decreased as the concentration increased. Fish pretreated with TPP exhibited significantly
decreased AchE activity whilst in the PBO pretreated group, increased activity was observed as compared with those exposed
to FNT alone. The pattern of AchE recovery was also assessed in fish previously exposed for 96h and then transferred to clean
(chemical free) water. Following 8 days of recovery period, the AchE activity of those exposed to FNT alone and pretreated
with TPP was still lower than that of the control. This study showed that FNT may cause hazard to fish after field application.
Organophosphorus insecticides (OPs) generally act through a common mechanism of toxicity initiated by inhibition of acetylcholinesterase (AChE). We studied the in vivo interactive toxicity of two common OPs, chlorpyrifos (CPF) and parathion (PS), in adult male rats. Dose–response studies estimated the acute oral LD1 values for the two OPs (CPF = 80 mg/kg po; PS = 4 mg/kg po) and these dosages or relative proportions were used to evaluate interactive toxicity. Three treatment strategies were evaluated: CPF followed by PS 4 h later (CPF-1st), PS followed by CPF 4 h later (PS-1st), and simultaneous (concurrent) exposures. Using LD1 dosages, rats in the CPF-1st and concurrent groups exhibited more cholinergic toxicity (i.e., salivation, lacrimation, urination, and diarrhea signs and involuntary movements) and higher lethality (7/8 and 6/8, respectively, beginning 1 h after PS) than those in the PS-1st group (2/8 lethality, beginning 3 days after CPF). Sequential exposures to lower dosages (CPF vs PS: 60 vs 3 mg/kg; 40 vs 2 mg/kg) led to more extensive neurotoxicity in the CPF-1st group compared to the other groups. Following lower dosages (40 vs 2 mg/kg), brain ChE inhibition was more extensive in the CPF-1st group at all time points (64–85%) and the concurrent group at 4 and 24 h after exposure (46–83%) compared to rats receiving PS first (7–48%). No differences were noted however, in plasma (71–93% inhibition) or liver (72–81%) cholinesterase activities nor were there group-related differences in plasma (50–60% inhibition) or liver (>85% inhibition) carboxylesterase activities. Incubation of liver samples with oxons in the presence or absence of calcium (i.e., 2 mM CaCl2 or EGTA) prior to addition of ChE (striatal sample) substantially blocked ChE inhibition by CPO (IC50: without liver = 4 nM; liver + calcium = 279 nM; liver + EGTA = 48 nM) but had lesser effects on PO-mediated inhibition (IC50: without liver = 17 nM; liver + EGTA = 56 nM; liver + calcium = 57 nM). Liver homogenate from animals preexposed to PS substantially decreased ChE inhibition by CPO when calcium was included (IC50: +EGTA = 8 nM; +calcium = 225 nM), but liver homogenate from animals preexposed to CPF was ineffective at blocking PO-induced inhibition (IC50: +EGTA = 16 nM; +calcium = 16 nM). We conclude that prior inhibition of carboxylesterase activity impacts toxicity of subsequent exposure to PS more than CPF because of more active detoxification of CPO by A-esterase. Together, these findings indicate that interactive toxicity from combined exposures to two OP insecticides can be markedly influenced by the sequence of administration.
Male and female rats were orally administered chlorpyrifos at a dose of 6.75 mg kg−1 body weight for 28 consecutive days. An additional chlorpyrifos group received zinc (227 mg l−1) in drinking water throughout the experimental duration. Two groups more served as controls; one received water only and the other received zinc in drinking water. Administration of chlorpyrifos resulted in a significant increase in lipid peroxidation (LPO) level and significant decrease in the activities of superoxide dismutase (SOD), glutathione-s-transferase (GST), catalase (CAT) and acetylcholinesterase (AChE) in erythrocytes of male and female rats. In contrast, zinc-chlorpyrifos treatment showed insignificant differences (p ⩽ 0.05–0.01), compared to control results, regarding LPO, SOD, GST and CAT. In case of AChE, supplementation of zinc showed little alteration in the activity of this enzyme in the rats treated with chlorpyrifos. It can deduce that chlorpyrifos induced oxidative stress and lipid peroxidation in erythrocytes of male and female rats. The overall results reveal the pronounced ameliorating effect of zinc in chlorpyrifos-intoxicated rats and variation in the response of male and female animals regarding alteration in the level of some biochemical parameters and LPO.
Biochemical systems in the mosquitofish were assayed in order to determine the factors contributing to the differences in toxicity among chlorpyrifos, parathion, and methyl parathion. In mosquitofish, chlorpyrifos was more toxic than parathion followed by methyl parathion. The brain cholinesterase (ChE) was more sensitive to chlorpyrifos-oxon than paraoxon followed by methyl paraoxon, assessed by the I50: 50nM for chlorpyrifos-oxon, 270nM for paraoxon, and 8400nM for methyl paraoxon. The muscle ChE was also more sensitive to chlorpyrifos-oxon than paraoxon followed by methyl paraoxon and was more sensitive to each compound than brain ChE; the I50's were: 6nM for chlorpyrifos-oxon, 60nM for paraoxon, and 540nM for methyl paraoxon. The hepatic aliesterases (AliE) were also more sensitive to chlorpyrifos-oxon than paraoxon followed by methyl paraoxon; the I50's were: InM for chlorpyrifos-oxon, 40nM for paraoxon, and 900nM for methyl paraoxon. Methyl parathion was activated by P450-mediated desulfuration in liver microsomes to the greatest extent followed by chlorpyrifos and parathion. Chlorpyrifos was detoxified by P450-mediated dearylation in liver microsomes more than parathion followed by methyl parathion. Hepatic A-esterases were capable of detoxifying chlorpyrifos-oxon, but had no significant effect on paraoxon or methyl paraoxon. The ChE sensitivity to oxon inhibition appears to reflect the toxicity levels of the parent insecticides, while metabolic factors are not predictions of toxicity levels.
Chlorpyrifos (O,O-diethylO-(3,5,6-trichloro-2-pyridinyl)-phosphorothioate) is an organophosphorus (OP) insecticide used for controlling insect pests. Currently, the reference dose (RfD) used by the Environmental Protection Agency (EPA) to establish acceptable human exposure tolerances for chlorpyrifos is based upon inhibition of blood butyrylcholinesterase (BuChE), which is not the target enzyme of chlorpyrifos, and does not play any role in cholinergic transmission. Data are presented showing that inhibition of acetylcholinesterase (AChE) associated with red blood cells (RBC), an enzyme similar to or identical with that in the nervous system, is a more appropriate endpoint on which to base the RfD. Basing an acceptable level of human exposure (e.g., RfD) on inhibition of RBC AChE provides a significant margin of safety, since it is 12- to 14-fold more sensitive as an indicator of chlorpyrifos exposure than the AChE in the most sensitive relevant neurological tissues (brain or retina). Inhibition of RBC AChE activity is consistently exhibited at lower dosages of chlorpyrifos than those required to result in clinical symptoms of OP toxicity, or alterations in cognitive functional responses. There is no unique sensitivity of the fetus or neonates to chlorpyrifos when administered by an appropriate oral dose. Thus, inhibition of RBC AChE activity is an appropriate surrogate measurement of chlorpyrifos exposure and provides a conservative endpoint for establishing appropriate margins of safety for both adults and infants.
Previous studies with rat liver perfused with Waymouth's medium have indicated that parathion is taken up extremely rapidly by hepatocytes of the periportal region of the lobule and undergoes “chromatographic” translobular migration. To determine whether this type of disposition takes place in vivo, two approaches were taken: (1) analysis of translobular behavior of pulse-infused parathion in the rat liver perfused with autologous blood in situ, and (2) autoradiographic demonstration of the tranlobular uptake pattern in vivo and in situ. To examine translobular behavior of parathion in the presence of blood, a recirculating autologous blood perfusion system was developed. Results were similar to those found with perfusion using artificial media. About 98–99% of a 0.4-ml pulse of 5 × 10−3M parathion was taken up by hepatocytes during the first pass and the fraction surviving the metabolism was eluted in a well-defined delayed peak. Autoradiographs from liver specimens obtained 4 hr after ip injections of 0.1, 0.8, or 2.0 mg/kg of [ethyl-1-3H]parathion showed dose-dependent exposure of intralobular zones. Low-dose (0.1 mg/kg) specimens showed radiolabeling within the first few rows of hepatocytes near portal vein branches whereas labeling in medium-dose (0.8 mg/kg) samples extended midway across the lobule with a sharp margin. In high-dose (2.0 mg/kg) autoradiographs, silver grains covered the entire lobule except for occasional small areas in the immediate vicinity of the central vein. Autoradiography of the liver pulse-infused with parathion in situ directly demonstrated that exposure to parathion/paraoxon was initially limited to the periportal region. These data established that the chromatographic pattern of translobular uptake and migration as observed previously in the liver perfusion with artificial media occurs in vivo and that exposure of centrilobular hepatocytes to parathion/paraoxon is dependent on the migration of parathion surviving metabolism after its initial periportal uptake.
Male rats were given a high sublethal dose of the organophosphate paraoxon (the potent anticholinesterase metabolite of the insecticide parathion) or a lethal dose of paraoxon antidoted with atropine to assure survival. These doses yielded a high level persistent inhibition of brain acetylcholinesterase, with 83–94% inhibition in the cerebral cortex, corpus striatum, and medulla oblongata within 2 hr of treatment, and still 25–45% inhibition 4 days after treatment. Recovery was faster in the medulla oblongata than the other two brain parts. Aging, as estimated by the amount of inhibition remaining after in vitro exposure to an oxime reactivator, gradually increased from no aging on the day of treatment to virtually complete aging at 4 days after treatment. Although a change in choline acetyltransferase activity could have helped compensate for the paraoxon-induced hypercholinergic activity to reduce overt symptomology and return other behaviors to normal levels, no paraoxon- or atropine-induced changes were observed in choline acetyltransferase specific activity in the cerebral cortex or corpus striatum at any time after treatment.
The ability of rat liver aliesterases to noncatalytically detoxify the oxons of six phosphorothionate insecticides was studied; the insecticides were methyl parathion, parathion, chlorpyrifos-methyl, chlorpyrifos, leptophos, and EPN. All oxons were more potent inhibitors (nM range) of rat liver aliesterases than the target rat brain acetylcholinesterase, with the exception of methyl paraoxon. Rat liver homogenates (including EDTA to eliminate possible A-esterase contributions) increased apparent I50s of the oxons to bovine brain acetylcholinesterase, indicating a detoxication of an appreciable amount of the oxon. Except for EPN-oxon, detoxication ability correlated with aliesterase sensitivity to inhibition. Liver homogenates from rats treated in vivo with the phosphorothionates had a reduced detoxication capability which correlated highly with residual aliesterase activity. With the exception of methyl parathion, animals treated for 90 min with high doses of the phosphorothionates displayed higher liver aliesterase inhibition than brain acetylcholinesterase inhibition. Thus, liver aliesterases represent a significant alternative phosphorylation site for organophosphates, and their efficacy for detoxication is a function of relative affinities of the oxon for the aliesterases and acetylcholinesterase.
Systemic exposure to parathion and paraoxon in normal and DDE-pretreated amle Sprague-Dawley rats was investigated by means of blood analyses following oral parathion doses and continuous parathion infusion into the perfused liver. Not only parathion, but paraoxon was also shown to undergo chromatographic translobular migration. As predicted from this, perfusion experiments, blood analyses, and autoradiography substantiated the existence of two discrete hepatic breakthrough thresholds for influent parathion, one for parathion and the other for paraoxon generated from parathion in the liver. In control rats, the former was slightly higher than the latter, resulting in systemic exposure to paraoxon without parathion exposure at sublethal toxic doses. Toxic symptoms correlated with the appearance of paraoxon in the jugular vein blood. In DDE-pretreated rats, both thresholds were substantially elevated, but enzyme induction had a greater impact on paraoxon breakthrough than on parathion release. Thus, significant amounts of parathion survived metabolism during migration through the lobules to appear in systemic blood, even at subtoxic doses. In contrast, paraoxon did not emerge from the liver under acute intoxication, although paraoxon was detected in the jugular vein. These results indicated that in DDE-pretreated rats paraoxon affecting the neural target was extrahepatically generated whereas the target is attacked by paraoxon originating in the liver in the control rats.
The oxidative desulfuration of the three phosphorothionate insecticides--chlorpyrifos, chlorpyrifos-methyl, and leptophos--was studied in rat brain and liver. Hepatic microsomes demonstrated activities of 4-28 nmol/g/min, with male activity 2- to 4-fold higher than female activity. Very low desulfuration activity of all three compounds was observed in both microsomal and crude mitochondrial fractions from brain (3-27 pmol/g/min). There were no sex differences in the brain. Although the liver displayed 140- to 2100-fold greater activity than brain on a wet-weight basis, the brain desulfuration activities of these three compounds as well as those of some previously reported phosphorothionates generally correlate well with the toxicity and may be important in determining the overall acute toxicity levels of phosphorothionate insecticides.
Cytochrome P-450-dependent monooxygenases are known to activate phosphorothionate insecticides to their oxon (phosphate) analogs by oxidative desulfuration. These activations produced potent anticholinesterases, decreasing the I50 values to rat brain acetylcholinesterase almost 1000-fold (from the 10(-5) M range to the 10(-8) M range). Since the usual cause of death in mammals from organophosphorus insecticide poisoning is respiratory failure resulting, in part, from a failure of the respiratory control center of the brain, we investigated the ability of rat brain to activate and subsequently degrade two phosphorothionate insecticides, parathion (diethyl 4-nitrophenyl phosphorothioate) and EPN (ethyl 4-nitrophenyl phenylphosphonothioate). Microsomes from specific regions (cerebral cortex, corpus striatum, cerebellum, and medulla/pons) of the brains of male and female rats and from liver were incubated with the phosphorothionate and an NADPH-generating system. Oxon production was quantified indirectly by the amount of inhibition resulting in an exogenous source of acetylcholinesterase added to the incubation mixture as an oxon trap. The microsomal activation specific activity was low for brain when compared to liver [0.23 to 0.44 and 5.1 to 12.0 nmol.min-1.(g tissue)-1 respectively]. The mitochondrial fraction of the brain possessed an activation activity for parathion similar to that of microsomes [about 0.35 nmol.min-1.(g tissue)-1 for each fraction], but mitochondrial activity was slightly greater than microsomal activity for EPN activation [0.53 to 0.58 and 0.23 to 0.47 nmole.min-1.(g tissue)-1]. Whole homogenates were tested for their ability to degrade paraoxon and EPN-oxon (ethyl 4-nitrophenyl phenylphosphonate), quantitated by 4-nitrophenol production. Specific activity for oxon degradation in liver was greater than that in brain [31 to 74 and 1.1 to 10.7 nmole.min-1.(g tissue)-1 respectively]. Overall, the brain and liver had about 1.5- to 12-fold higher specific activities for degradation than activation depending on the compound used. These findings demonstrate that the brain possesses both phosphorothionate activation and oxon degradation abilities, both of which may be significant during exposures to organophosphorus insecticides.
The ability of phenobarbital and beta-naphthoflavone to induce parathion desulfuration, aminopyrine N-demethylation, and NADPH-cytochrome-c reductase activity in the brain and liver of male and female rats was investigated. Activities of all three enzymes were found in similar levels in both the mitochondrial and microsomal fractions of brain. There were no sex differences in brain activities. Liver activities were from 10- to 30-fold higher than brain activities when computed on a tissue-wet-weight-equivalent basis. Although exposure to both inducers increased all three enzyme activities and cytochrome P-450 in liver, neither inducer increased the enzyme activities in mitochondrial or microsomal brain fractions of either sex. Thus, these brain monooxygenase activities appear to be refractory to induction by two classical types of cytochrome P-450 inducers. This lack of inducibility could serve to protect the animal against environmentally enhanced increases in the activation of xenobiotics to neurotoxic metabolites, such as parathion desulfuration to paraoxon.
The dimethyl-substituted organothiophosphate insecticides methyl parathion and azinphos-methyl are thought to undergo glutathione-mediated detoxification in mammals. In the present study, depletion of hepatic glutathione in the mouse by pretreatment with diethyl maleate potentiated the acute toxicities of methyl parathion and azinphos-methyl, whereas depletion of hepatic glutathione by pretreatment with buthionine sulfoximine did not. Furthermore incubation of 50 microM methyl parathion with mouse hepatic microsomes for 5 min in the presence of 1 mM diethyl maleate led to significantly greater (p less than 0.05) production of methyl paraoxon, compared to incubations in the absence of diethyl maleate. Conversely, 1 mM diethyl maleate had no effect on metabolic activation of azinphos-methyl by mouse hepatic microsomes, while 10 mM inhibited slightly production of azinphos-methyl oxon from azinphos-methyl. These results suggest normal levels of hepatic glutathione are not required for detoxification of methyl parathion or azinphos-methyl in the mouse. Moreover the potentiation of the acute toxicity of methyl parathion following diethyl maleate pretreatment could result, at least in part, from enhanced production of methyl paraoxon. However, diethyl maleate likely acts through another mechanism(s) as well since it did not enhance the metabolic activation of azinphos-methyl in vitro. These data raise serious doubts about the participation of glutathione in the detoxification of methyl parathion and azinphos-methyl in vivo in the mouse.
The specific activities of hepatic enzymes involved in both the activation and detoxification of the anticholinesterase insecticide parathion were significantly greater in both rats and mice compared to either fathead minnows or rainbow trout. Whereas the rates of parathion desulfuration and hydrolysis were comparable, the specific activity for paraoxon hydrolysis was approximately threefold greater than its rate of formation for all species except trout. The kinetic data, however, indicate that the significantly greater Km for paraoxon hydrolysis limits its detoxification. Accordingly, the data are consistent with the progressive accumulation of this toxic metabolite in liver tissue incubated in vitro with parathion. The similar Km for parathion desulfuration and hydrolysis by rodents is consistent with the proposed mechanism of catalysis involving a common S-oxide intermediate. Due to the slow rates of enzyme catalysis, reliable kinetic estimates for the hydrolytic reactions were not possible for both species of fish. Assuming comparable Km values for paraoxon hydrolysis in rodents and fish, the net accumulation of paraoxon in tissue water may be quite similar between species and metabolic transformation may not be an important determinant of the species-selective toxicity of parathion.
Single-pass perfusion of mouse livers in situ with the phosphorothioate pesticide parathion resulted in formation of the cholinesterase inhibitor paraoxon (PO), p-nitrophenol (PNP), p-nitrophenyl sulfate (PNPS), and p-nitrophenyl glucuronide (PNPG). Daily pretreatment of mice with phenobarbital (80 mg/kg, ip) for 4 days induced hepatic cytochrome P-450 content, as well as oxidative activation and oxidative detoxification of parathion, as measured in vitro. However, phenobarbital pretreatment did not alter production of PO from parathion in mouse livers perfused in situ, although it increased production of PNP, PNPS, and PNPG. Additionally, phenobarbital pretreatment antagonized the acute toxicity of parathion in mice. These results indicate that phenobarbital pretreatment clearly induces that form(s) of cytochrome P-450 catalyzing conversion of parathion to PO. Yet increased amounts of PO do not exit perfused livers from phenobarbital pretreated mice. Instead, the enhanced detoxification of parathion to PNP, PNPS, and PNPG likely results in the observed antagonism of parathion's acute toxicity.
5 cytochrome P-450 isozymes were purified from the livers of uninduced mice and reconstituted with purified NADPH cytochrome P-450 reductase and phospholipid. The pesticides parathion, fonofos, DEF, Mocap and profenofos were oxidized by the reconstituted monooxygenase system to form acetylcholinesterase (AChE) inhibitors. The bioactivation varied with the pesticide substrate and the cytochrome P-450 isozyme. Aldrin epoxidation occurred with all 5 isozymes, with cytochrome P-450 A1 being the most active. All fraction metabolized the pesticide synergist piperonyl butoxide (PBO) to form an inhibitory cytochrome P-450-PBO-metabolite complex. The reduced complex produced a spectrum in the Soret region which was characteristic for each of the cytochrome P-450 isozymes. Inhibition of aldrin epoxidation by PBO was found to be unrelated to the nature of the Soret spectrum.
1. The metabolism of the phosphorothionate parathion in vitro was examined by using [(32)P]parathion and microsomes isolated from the livers of various animal species. 2. The major metabolic products of parathion in this system in vitro were identified as diethyl 4-nitrophenyl phosphate (paraoxon), diethyl hydrogen phosphate, diethyl hydrogen phosphorothionate and p-nitrophenol. 3. The reaction leading to the formation of diethyl hydrogen phosphorothionate and p-nitrophenol requires the same cofactors (NADPH and oxygen) required for metabolism of parathion to its active anti-acetylcholinesterase paraoxon. 4. The enzyme activity towards parathion per unit weight of liver is increased some 65-130% by pretreatment of male rats with phenobarbital and 3,4-benzopyrene. 5. The metabolism of parathion is inhibited by incubation in a nitrogen atmosphere and in an atmosphere containing carbon monoxide. Pure oxygen is also inhibitory. These results are discussed in terms of a deficiency of oxygen for maximal activity as well as the lability of some component of the system to oxidation.
Rats were administered high sublethal intraperitoneal dosages of the phosphorothionate insecticides parathion, methyl parathion, and chlorpyrifos, and their oxons. Acetylcholinesterase activities in cerebral cortex and medulla oblongata and aliesterase activities in liver and plasma were monitored at 2 hr and 1, 2, and 4 days after exposure. The maximal inhibition of brain acetylcholinesterase activity was not immediate with parathion and chlorpyrifos, reflecting the time required for bioactivation of the phosphorothionates as well as the effectiveness of the aliesterases to inactivate much of the hepatically generated oxons. In contrast, brain acetylcholinesterase activities were more quickly inhibited following administration of paraoxon and chlorpyrifos-oxon, which do not require bioactivation. Brain acetylcholinesterase was also rapidly inhibited following administration of methyl parathion and methyl paraoxon, reflecting the low sensitivity of the aliesterases to methyl paraoxon. Aliesterases were inhibited to a greater extent than acetylcholinesterase at each sampling time with parathion and chlorpyrifos and their oxons, whereas the reverse was true with methyl parathion and methyl paraoxon. All of the above patterns correlate with the in vitro sensitivities of acetylcholinesterase and aliesterases to the oxons. The very prolonged inhibition of esterase activities following chlorpyrifos treatment probably results from its substantially greater lipophilicity compared to the other compounds, which would allow it to be stored and released for gradual bioactivation. The data reported indicate that the disposition and effects of different phosphorothionate insecticides will be influenced by the sensitivities of target and nontarget esterases for their oxons and by their lipophilicity, and that predictions of in vivo responses can be made from in vitro data.
1.1. LD50 values for 42 pesticides and 2 metabolites of DDT administered in a single dose by the oral or dermal route to Sherman strain adult rats have been determined.2.2. A comparison of laboratory results and use experience indicates that there is a much closer relationship between dermal LD50 values and the occurrence of occupational poisoning than between oral LD50 values and occupational poisoning.3.3. The carbamate pesticide Isolan was observed to be different from other compounds tested in that it was considerably more toxic by the dermal route than by the oral route to rats.