Oxon Forms Research Articles

Activated transformations of organophosphorus insecticides in the case of non-AChE inhibitory oxons.

Many organophosphorus (OP) compounds are of the thiono form and in insects or animals are converted by microsomal mixed function oxidases (MFO) into the oxon forms which inhibit acetylcholinesterase (AChE) and give toxic activity. However, certain S-alkyl phosphorothiolates (RS-P(O) <) such as methamidophos, profenophos and prothiophos oxon are strongly insecticidal, but very poor inhibitors of AChE in vitro. Their oxons are converted further to the S-oxides, which either inhibit AChE or decompose, depending on the alkyl substituents on the sulfur atom. It is also inferred in the case of prothiophos oxon that its S-oxide not only inhibits AChE but also conjugates with glutathione (GSH) by the action of glutathione S-transferase (GST), and the conjugate inhibits AChE. Certain phosphoramidates (R2N-P(O) <) such as isofenphos oxon, schradan and propetamphos oxon are weak AChE inhibitors, but strongly insecticidal. It is well known that isofenphos oxon is converted into the stable N-desalkyl form (H2N-P(O) <) by oxidative dealkylation to inhibit AChE. The authors have studied activation of phosphoramidates using 2,4-dichlorophenyl methyl N-alkylphosphoramidates as model compounds using various approaches including computational chemistry, and these studies indicated that the O-aminophosphate structure (R2N-O-P(O) <) is an activated form.

Kinetic parameters of OPT pesticide desulfuration by c-DNA expressed human CYPs

The role of different cytochrome P450 isoforms (CYPs) in the desulfuration of four organophosphorothionate pesticides (OPTs), namely diazinon (DIA), azinphos-methyl (AZ), chlorpyrifos (CPF) and parathion (PARA), at OPT levels representative of actual human exposure has been investigated. For this purpose c-DNA expressed human CYPs and a method, based on acetylcholinesterase (AChE) inhibition, able to detect nM levels of oxon have been used. Our results indicate that the four tested OPTs at low concentration were mainly desulfurated by CYP2B6, 2C19 and 1A2, showing K m values in the range 0.8–5 μM and the highest efficiency (intrinsic clearance (ICL)) values. CYP3A4 was generally endowed with high K m and resulted linear up to 25–100 μM OPT, concentrations saturating the most efficient CYPs. The tentative extrapolation of the relative contribution of single CYPs, taking into account the average content of different isoforms in the human liver, indicate that CYP1A2 is the major responsible for oxon formation. Indeed this CYP catalyses the 50–90% of desulfuration reaction, depending on the OPT. As CYP3A4 activity is not completely saturated up to 100 μM OPT, and due to the high hepatic content, its contribution to oxon formation may result relevant in poisoning episodes, when individuals are exposed at high doses of OPTs.

INHIBITION OF CHOLINESTERASE ENZYMES FOLLOWING A SINGLE DERMAL DOSE OF CHLORPYRIFOS AND METHYL PARATHION, ALONE AND IN COMBINATION, IN PREGNANT RATS

Pregnant Sprague-Dawley rats (14-18 d of gestation) were treated with either a single dermal subclinical dose of 30 mg/kg (15% of dermal LD50) chlorpyrifos (O,O-diethyl O-[3,5,6-trichloro-2-pyridinyl] phosphorothioate) or a single dermal subclinical dose of 10 mg/kg (15% of dermal LD50) methyl parathion (O,O-dimethyl O-4-nitrophenyl phosphorothioate) or the two in combination. Chlorpyrifos inhibited maternal and fetal brain acetylcholinesterase (AChE) activity within 24 h of dosing, (48% and 67% of control activity, respectively). Following application of methyl parathion, peak inhibition of maternal and fetal brain AChE activity occurred at 48 h and 24 h after dosing (17% and 48% of control activity, respectively). A combination of chlorpyrifos and methyl parathion produced peak inhibition of maternal and fetal brain AChE activity at 24 h postdosing (35% and 73% of control activity, respectively). Maternal and fetal brain AChE activity recovered to various degrees of percentage of control 96 h after dosing. Application of methyl parathion or chlorpyrifos alone or in combination significantly inhibited maternal plasma butyrylcholinesterase (BuChE) activity. No significant inhibition of fetal plasma BuChE activity was detected. Peak inhibition of maternal liver BuChE occurred 24 h after application of methyl parathion or chlorpyrifos alone or in combination (64%, 80%, and 61% of control activity, respectively). Significant inhibition of placental AChE occurred within 24 h after application of methyl parathion or chlorpyrifos alone or in combination. The results suggest that methyl parathion and chlorpyrifos, alone or in combination, were rapidly distributed in maternal and fetal tissues, resulting in rapid inhibition of cholinesterase enzyme activities. The lower inhibitory effect of the combination could be due to competition between chlorpyrifos and methyl parathion for cytochrome P-450 enzymes, resulting in inhibition of the formation of the potent cholinesterase inhibitor oxon forms. The faster recovery of fetal plasma BuChE is attributed to the de novo synthesis of cholinesterase by fetal tissues compared to maternal tissues.

Automated detection of chlorpyrifos and its metabolites by a continuous flow system-based enzyme sensor

This work is a continuation of the development of a previously described pesticide sensor and presents significant improvements including automation of the system and a new cell design [Biosens. Bioelectron. 13 (1998) 213]. These modifications to the continuous flow system permitted a reduction of analysis time and led to a better reproducibility of the delivery times of the different reagent solutions. Chlorpyrifos, methyl-chlopyrifos and their metabolites (oxon forms and trichloro pyridinol) were used for testing the continuous flow system. Kinetics using free acetylcholinesterase activity showed that pesticides were one 1000-fold less toxic than the oxon forms (bimolecular rate constants ( k i) for chlorpyrifos-oxon and methyl-chlorpyrifos-oxon were 3.0×10 6 and 1.3×10 6 M −1 min −1, respectively). Trichloro-pyridinol did not inhibit free acetylcholinesterase activity. In continuous flow conditions, chlorpyrifos, methyl-chlopyrifos and trichloropyridinol did not significantly decrease the activity of the immobilised enzyme. The biosensor detection of chlorpyrifos-oxon gave a calibration curve with a lowest detectable concentration ranging from 0.72×10 −6 M (240 ppb) to 73×10 −9 M (24 ppb) when the incubation time was increased from 1 to 16 min. The rinsing step after biosensor inhibition by methyl-chlorpyrifos-oxon led to a partial reactivation of the immobilised enzyme. Under these conditions, the lowest detectable concentration ranged from 2.3×10 −6 M (700 ppb) to 0.89×10 −6 M (270 ppb) when the incubation time was increased from 1 to 8 min.

Analysis of paraoxonase (PON1) L55M status requires both genotype and phenotype.

Paraoxonase (PON1) is tightly associated with high-density lipoprotein particles and is believed to contribute to the prevention of atherosclerosis by metabolizing oxidized lipids. PON1 also hydrolyses the bioactive oxon forms of organophosphorus pesticides such as parathion, diazinon and chlorpyrifos. Two common polymorphisms have been identified in the coding sequence of human PON1: L55M and R192Q. Several previous studies have found that the presence of the PON1R192 allele raises the risk of cardiovascular disease while others found no correlation. The studies, however, have focused on the genotype of PON1 and not the expression level of the protein. We found that the PON1 expression level in plasma, as determined by the rates of paraoxon and diazoxon hydrolysis, varies widely among individuals and within a genotype. Previous studies found that individuals having Met at PON155 have lower levels of both PON1 mRNA and activity. In this study, we determined the plasma activity levels of PON1 and examined the relationships between PON155 genotype and PON1 level. As with PON1192, we found considerable overlap in activity among the PON155 genotypes. Of the 317 individuals whose PON1 status was determined in this study, 48.9% were PON1Q192 homozygotes. Analysis of the PON1QQ192 population showed that while the average PON1 activity (diazoxon hydrolysis) was 12266 U/L for PON1LL55 and 7777 U/L for PON1MM55, a given PONMM55 individual could have more than twice the activity of a PON1LL55 individual. PON1 status, which includes PON1 level as well as PON1192 genotype, may be a better predictor for cardiovascular disease or organophosphate susceptibility than PON1 genotype alone.

Fate of Summertime Airborne Organophosphate Pesticide Residues in the Sierra Nevada Mountains

AbstractThis study examines the processes of dilution, degradation, and sorption to plant foliage of organophosphate (OP) pesticides during the summertime in an air corridor originating in the southern Central Valley of California and moving into the nearby Sierra Nevada mountains. Residues of chlorpyrifos, methidathion, and their oxons were examined in air and pine needles at three sites in the southern Sierra to delineate the role these processes play in the atmospheric fate of these residues. At the site closest to the Central Valley, we found relatively high levels of parent OPs and oxons in needle and air samples. At higher elevations needles contained lesser amounts of OP residues and at lower frequency, while air primarily contained the oxon form. With increasing elevation the ratio of thion to oxon form of chlorpyrifos in air decreased from 1.85 to 0.46 indicating that atmospheric oxidation was occuring. Based on the amounts of foliar deposition found, we estimate that during summer months nearly 16 kg of chlorpyrifos and its oxon may enter Sequoia National Park plant foliage. We deduce that for airborne OP insecticides, foliar deposition is a significant summertime fate process, along with atmospheric degradation and dilution.

The effect of the human serum paraoxonase polymorphism is reversed with diazoxon, soman and sarin.

Many organophosphorus compounds (OPs) are potent cholinesterase inhibitors, accounting for their use as insecticides and, unfortunately, also as nerve agents. Each year there are approximately 3 million pesticide poisonings world-wide resulting in 220,00 deaths. In 1990, there were 1.36 million kg of chlorpyrifos, 4.67 million kg of diazinon and 1.23 million kg of ethyl parathion manufactured in the USA (data supplied by the USEPA). In addition to exposure risks during pesticide manufacturing, distribution and use, there are risks associated with the major international effort aimed at destroying the arsenals of nerve agents, including soman and sarin. The United States has pledged to destroy approximately 25,000 tons of chemical agents by the end of the decade. The high density lipoprotein (HDL)-associated enzyme paraoxonase (PON1) contributes significantly to the detoxication of several OPs (Fig. 1). The insecticides parathion, chlorpyrifos and diazinon are bioactivated to potent cholinesterase inhibitors by cytochrome P-450 systems. The resulting toxic oxon forms can be hydrolysed by PON1, which also hydrolyses the nerve agents soman and sarin (Fig. 1). PON1 is polymorphic in human populations and different individuals also express widely different levels of this enzyme. The Arg192 (R192) PON1 isoform hydrolyses paraoxon rapidly, while the Gln192 (Q191) isoform hydrolyses paraoxon slowly. Both isoforms hydrolyse chlorpyrifos-oxon and phenylacetate at approximately the same rate. The role of PON1 in OP detoxication is physiologically significant. Injected PON1 protects against OP poisoning in rodent model systems and interspecies differences in PON1 activity correlate well with observed median lethal dose (LD50) values. We report here a simple enzyme analysis that provides a clear resolution of PON1 genotypes and phenotypes allowing for a reasonable assessment of an individual's probable susceptibility or resistance to a given OP, extending earlier studies on this system. We also show that the effect of the PON1 polymorphism is reversed for the hydrolysis of diazoxon, soman and especially sarin, thus changing the view of which PON1 isoform is considered to be protective.

Prediction of inter-species differences in the toxicity of organophosphorus pesticides to wildlife—a biochemical approach

The activation of organophosphorus pesticides and sensitivity of ‘B’ esterases to inhibition (I 50) by the oxon metabolites were investigated in brain, liver and serum as the basis for a model to predict the toxicity of organophosphorus pesticides to four avian species. There were statistically significant correlations between LD 50 and brain rate of activation of the OP ( r = 0.68), brain acetylcholinesterase I 50 ( r = 0.88), and serum carboxylesterase I 50 ( r = 0.60). A significant proportion of the oxon produced within the liver is unlikely to reach the brain, due to irreversible binding by ‘B’ esterases. The production of the active oxon form of the pesticide within the brain, and the sensitivity of the brain AChE to inhibition, are probably the most important factors in determining the avian toxicity of organophosphorus pesticides.

List of journals scanned

This work is a continuation of the development of a previously described pesticide sensor and presents significant improvements including automation of the system and a new cell design [Biosens. Bioelectron. 13 (1998) 213]. These modifications to the continuous flow system permitted a reduction of analysis time and led to a better reproducibility of the delivery times of the different reagent solutions.Chlorpyrifos, methyl-chlopyrifos and their metabolites (oxon forms and trichloro pyridinol) were used for testing the continuous flow system. Kinetics using free acetylcholinesterase activity showed that pesticides were one 1000-fold less toxic than the oxon forms (bimolecular rate constants (ki) for chlorpyrifos-oxon and methyl-chlorpyrifos-oxon were 3.0×106 and 1.3×106 M−1 min−1, respectively). Trichloro-pyridinol did not inhibit free acetylcholinesterase activity.In continuous flow conditions, chlorpyrifos, methyl-chlopyrifos and trichloropyridinol did not significantly decrease the activity of the immobilised enzyme. The biosensor detection of chlorpyrifos-oxon gave a calibration curve with a lowest detectable concentration ranging from 0.72×10−6 M (240 ppb) to 73×10−9 M (24 ppb) when the incubation time was increased from 1 to 16 min. The rinsing step after biosensor inhibition by methyl-chlorpyrifos-oxon led to a partial reactivation of the immobilised enzyme. Under these conditions, the lowest detectable concentration ranged from 2.3×10−6 M (700 ppb) to 0.89×10−6 M (270 ppb) when the incubation time was increased from 1 to 8 min.

Interactive effects between EBI fungicides (prochloraz, propiconazole and penconazole) and OP insecticides (dimethoate, chlorpyrifos, diazinon and malathion) in the hybrid red-legged partridge

The toxicokinetic interactions between the ergosterol-biosynthesis-inhibiting (EBI) fungicides prochloraz, propicon-azole, and penconazole and the organophosphorus (OP) insecticides dimethoate, chlorpyrifos, and diazinon have been studied in the hybrid red-legged partridge. The inhibition of serum butyrylcholinesterase (BuChE) activity provided a useful biochemical indicator of the generation of the toxic oxon metabolites of these OP insecticides. Birds pretreated with 180 mg/kg prochloraz tended to show a greater inhibition of serum BuChE activity at 1, 4, and 24 h following oral exposure to either of the OPs dimethoate (3 mg/kg) or chlorpyrifos (9 mg/kg) compared to birds pretreated with corn oil. Prochloraz-pretreated birds also showed a tendency toward an increased inhibition at 24 h following dosing with the OP diazinon (4.3 mg/kg) compared to corn oil controls. In the case of dimethoate, the inhibition of serum BuChE activity was significantly greater in treated birds than controls at 24 h. Birds pretreated with the EBI fungicide propiconazole (200 mg/kg) showed a similar inhibition of serum BuChE activity to those pretreated with corn oil following administration of 167 mg/kg malathion. Pretreatment with the EBI fungicide penconazole (200 mg/kg) produced significantly greater depression of serum BuChE activity at 1, 4, and 24 h after dosing with malathion, when compared to corn oil controls. The tendency toward increased inhibition of serum BuChE activity by each of the OPs in prochloraz-pretreated birds was attributed to an increased activation of the compound to its active oxon form as a consequence of induction of microsomal monooxygenases by prochloraz.

Avian serum esterases: Species and temporal variations and their possible consequences

Avian serum esterases are predominantly of the ‘B’ type (cholinesterases and carboxylesterases) and are inhibited by carbamates and the ‘active’ oxon forms of organophosphorus pesticides. Selective inhibition of mammalian serum carboxylesterase, a ‘B’ esterase, has shown that this enzyme may play an important role in detoxication by irreversibly binding, and thus inactivating, anticholinesterase compounds. Studies have shown differences between carnivorous and omnivorous/herbivorous avian species in the level of activity and range of forms of carboxylesterases and cholinesterases in sera. In addition, these enzymes show seasonal, diurnal and developmental variations in activity. This paper will discuss species and temporal variations in avian serum ‘B’ esterases in relation to their possible influence on pesticide toxicity.

Degradation of [14C]isofenphos in soil in the laboratory under different soil pH's, temperatures, and moistures.

The effects of three soil pH's, three soil temperatures, and three soil moistures on [14C]isofenphos degradation were investigated. All three factors interacted strongly and significantly affected the persistence of isofenphos as well as the formation of the degradation products (p less than 1%). Isofenphos degradation was greatest at the higher temperatures 35 degrees C greater than 25 degrees C greater than 15 degrees C (except under alkaline pH's), medium moisture 25% greater than 30% greater than 15%, and in both alkaline (pH = 8) and acidic soils (pH = 6) compared with neutral soil (pH = 7). Isofenphos oxon formation was greatest at higher temperatures 35 degrees C compared with 25 degrees C and 15 degrees C, in acidic soil greater than neutral soil greater than alkaline soil, and under high moisture (30%) compared with the 15% and 22.5% moistures. The formation of soil-bound residues was greatest at higher temperatures 35 degrees C greater than 25 degrees C greater than 15 degrees C, higher moisture 30% compared with 15% and 22.5%, and in alkaline soil compared with neutral and acidic soils.

Transformation of DAEP (S-(2-acetylaminoethyl) dimethyl phosphorodithioate) under various oxidative conditions.

14C-DAEP [S-(2-acetylaminoethyl) dimethyl phosphorodithioate] was subjected to four different oxidative conditions, and the products were identified. On peracid oxidation in dichloromethane, DAEP gave the oxon (1) predominantly, and 2-acetylaminoethyl dimethoxyphosphinyl disulfide (3), N-acetylcysteamine (10), its oxidized dimer (11) and a further oxidation product of compound 11. This indicates that an unstable phosphorus oxythionate was initially formed, which lost sulfur, was rearranged, and hydrolyzed to give these products. Under other conditions, phosphinyl disulfide 3 was not found. DAEP was metabolized in vitro with a rat liver microsome-NADPH system via oxidation. The aqueous reaction condition prevented the formation of compound 3 from the intermediate, which predominated as well as the oxon formation under anhydrous or close conditions. The formation of various products with sunlight irradiation on glass plates or on bean leaves could be interpreted by oxidation at P=S, C1 and C2 positions, demethylation, and deacetylation, followed by further transformation. The initial formation of phosphorus oxythionate seems to play an important role in the oxidation of the organothionophosphorus compound.

Esterase inhibition and reactivation in relation to piperonyl butoxide-phosphorothionate interactions

The effect of piperonyl butoxide on the acute toxicity of phosphorothionate insecticides was studied in male mice. One hour after piperonyl butoxide (400 mg/kg), the toxicity of the dimethyl phosphorothionates, methyl parathion and Guthion, was antagonized, whereas the toxicity of their respective diethyl homologs, parathion and Ethyl Guthion, was potentiated. Piperonyl butoxide did not appreciably alter the toxicity of the oxygen analogs of these compounds. Pretreatment with SKF 525-A (50 mg/kg) modified the toxicity of the phosphorothionates in a manner qualitatively similar to piperonyl butoxide pretreatment. Plasma concentrations of all four insecticides were increased three- to sevenfold in piperonyl butoxide-pretreated mice. This increase may result in a greater total oxon formation; however, reactivation in vitro of esterases inhibited in vivo was 5 to 10 times more rapid following methyl parathion or Guthion challenge than after their diethyl homologs. Although a greater total oxon formation-cholinesterase inhibition is possible for both dimethyl and diethyl phosphorothionates following piperonyl butoxide pretreatment, rapid reactivation of inhibited nerve tissue cholinesterases after dimethyl phosphorothionate challenge appears to compensate for further inhibition occurring at a decreased rate. The net result would be a reduction in dimethyl phosphorothionate toxicity. In contrast, slow reactivation of inhibited nerve tissue cholinesterases following diethyl phosphorothionate challenge appears unable to compensate for increased oxon formation-cholinesterase inhibition. The net result is a potentiation of the toxicity of the diethyl-substituted compounds.