Analysis of the additivity of in vitro inhibition of cholinesterase by mixtures of chlorpyrifos-oxon and azinphos-methyl-oxon.

Organophosphorus (OP) insecticides or their active metabolites act through a common mechanism of toxicity, the inhibition of cholinesterase (ChE). The effects of in vitro exposure of brain (target) and serum (biomarker) ChE to chlorpyrifos-oxon (C horizontal lineO) and azinphos-methyl-oxon (AZM horizontal lineO), the active metabolites of the insecticides chlorpyrifos and azinphos-methyl, respectively, were investigated to determine if simultaneous or sequential exposure to these two OP compounds results in purely additive effects. Additive was defined by the theoretical calculated percent inhibition (dose additivity), which takes into account the fraction of ChE molecules assumed to be available for inhibition by the second compound following inhibition by the first compound, not simple mathematical summation of percent inhibition (response additivity). Brain ChE simultaneously exposed to the two compounds resulted in additive effects, which were less than the simple mathematical summation of percent inhibition. However, serum ChE simultaneously exposed to the two compounds resulted in a nonlinear response, presumably due in part to the presence of detoxifying enzymes in the serum. Sequential exposure of both brain and serum ChE to the two compounds resulted in greater than additive effects at the higher concentrations of each compound. There was no departure from additivity at the lower concentrations of the two compounds. These data suggest that simple mathematical summation of percent inhibitions, i.e., response additivity, is not the appropriate method for describing the combined effects of C horizontal lineO and AZM horizontal lineO on ChE in vitro. In addition, there are other mechanisms involved, such as the presence of detoxication enzymes, that must be taken into account when analyzing the effects of combined exposure of ChE to these two compounds.

Effects of repeated oral postnatal exposure to chlorpyrifos on open-field behavior in juvenile rats.

Organophosphorus (OP) insecticides have the potential to cause behavioral effects in children. This study was designed to determine if repeated oral exposure of preweanling rats to chlorpyrifos would produce behavioral changes at both pre- and postweanling ages. Treatment occurred every second day beginning on post-natal day (PND) 1, and continued through PND 21. The rats received one of the following regimens: a low-dosage (3 mg/kg) from PND 1-21; a medium dosage (mg/kg from PND 1-5, and then 6 mg/kg from PND 7-21; or a high-dosage schedule of 3 mg/kg on PND 1-5, then 6 mg/kg from PND 7-13, and 12 mg/kg from PND 15-21. There were no differences in body weights among the control-, low-, and medium-dosage groups but the high-dosage group had significantly lower body weights on PND 13-21. An open field was used to measure locomotor activity on PND 10, 12, 14, 16, 18, 20, 25, and 30. There were no differences in locomotor activity levels or treatment effects between males and females. On PND 10, 12, 14, 16, 18, and 20 there was no effect on locomotor activity with any dosage. On days 25 and 30, locomotor activity was significantly decreased with the medium- and high-dosage groups. Brain cholinesterase (ChE) inhibition was about 25-38% on PND 25 and 14-34% on PND 30. On PND 25 but not 30, lung and diaphragm ChE and serum butyrylcholinesterase (BChE), with the high-dosage animals, and heart ChE with the medium- and high-dosage groups were significantly inhibited. There was no significant inhibition of skeletal muscle ChE or serum acetylcholinesterase (AChE) on PND 25 and 30. These data suggest that early postnatal chlorpyrifos exposures will depress locomotor activity in juvenile rats, with the effects most pronounced after brain ChE activity has substantially recovered.

Purification of two rat hepatic proteins with A-esterase activity toward chlorpyrifos-oxon and paraoxon.

A-esterases are calcium-dependent hydrolases that can detoxify the active metabolites (oxons) of organophosphorus insecticides such as chlorpyrifos and parathion. A-esterases from rat liver have previously been shown to hydrolyze chlorpyrifos-oxon but not paraoxon at low substrate concentrations. Two A-esterases were extracted by ammonium sulfate fractionation from solubilized rat liver microsomes followed by gel filtration chromatography and preparative scale isoelectric focusing. The proteins displayed similar characteristics and were difficult to separate; both had similar high molecular mass and isoelectric point range and exhibited A-esterase activity toward high and low concentrations of chlorpyrifos-oxon and high concentrations of paraoxon. Sufficient amounts of the higher molecular mass protein were obtained for kinetic studies, which yielded a Km of 0.93 mM toward high concentrations of chlorpyrifos-oxon and a Vmax of 369 nmoles product formed/mg protein-min. The protein hydrolyzed phenyl acetate, chlorpyrifos-oxon and paraoxon, suggesting that arylesterase and A-esterase activities are attributable to the same liver protein(s). Assays of purified protein and kinetic studies of microsomes suggested that the activity toward high (320 microM) and low (

Identification and isolation of two rat serum proteins with A-esterase activity toward paraoxon and chlorpyrifos-oxon.

The active metabolites (oxons) of phosphorothionate insecticides can be detoxified via A-esterase hydrolysis. Two enzymes with A-esterase activity have been isolated from rat serum. Whole serum was applied to anion exchange gel (DEAE Sepharose Fast Flow) and incubated (1 hr). Tris-HCl buffer (0.05 M; pH 7.7, at 5 degrees) containing 0.25 M NaCl was added to the slurry and incubated. The decant, containing low A-esterase activity but a high protein concentration, was discarded. Further displacement of A-esterase from DEAE gel was achieved with 1.0 M NaCl in 0.05 M Tris-HCl buffer (Ph 7.7 at 5 degrees). Following desalting and concentration, further separation was achieved by gel filtration (Sephacryl S-100 HR) and two sequential preparative scale isoelectric focusings. Final fractions contained two proteins of high molecular mass (one about 200 kDa and one between 137 and 200 kDa). The apparent range of isoelectric points for the two enzymes was 4.5 to 5.6. Following native-PAGE analysis, activity stains with beta-naphthyl acetate and Fast Garnet GBC in the presence of paraoxon (10-5 M) verified that A-esterase activity was associated with both proteins. Spectropho-tometric assay detected A-esterase activity toward paraoxon, chlorpyrifos-oxon, and phenyl acetate in the final preparation.

Organophosphate detoxication potential of various rat tissues via A-esterase and aliesterase activities.

Paraoxon and chlorpyrifos-oxon, active metabolites of the organophosphorus insecticides parathion and chlorpyrifos, can be detoxified via A-esterases and aliesterases. These enzyme activities were measured in various tissues of Sprague-Dawley rats. High A-esterase activities were detected in liver, serum and liver mitochondrial/microsomal fractions. Low or no A-esterase activities were detected in other tissues and tissue fractions. A-Esterase substrate:substrate activity ratios suggest that the substrates are probably not degraded by the same enzyme. Highest aliesterase activities were observed in the small intestine and liver with moderate activity in kidney, serum and lungs. Low activities were noted in brain, spleen and skeletal muscle.

A comparative study of inhibition of acetylcholinesterase, trypsin, neuropathy target esterase, and spleen cell activation by structurally related organophosphorus compounds.

Organophosphorus (OP) compounds can bind to and inactivate several target molecules other than acetylcholinesterase (AChE). In the present study, five sets of structurally related organophosphorus compounds were used to evaluate the relationships between organophosphorus binding sites of AChE, neuropathy target esterase (NTE), trypsin, and the target molecule(s) involved in inhibition of splenocyte activation by OP compounds. The concentration of each OP compound required to inhibit enzyme activity or splenocyte activation by concanavalin A by 50% was determined. The pattern of IC50 values indicated that AChE, trypsin, NTE, and the molecule(s) involved in inhibition of splenocyte activation are distinct with regard to patterns of inhibition by OP compounds. However, there was a striking similarity in the patterns of inhibition for trypsin and NTE with substantial differences for only 2 of 20 compounds. This pattern suggests similarity in the active sites of these molecules. There were also similarities in the IC50 patterns for lymphocyte activation and trypsin or NTE activity. However, the correlation was not as strong as between NTE and trypsin, and the data suggested the possibility of multiple target molecules for inhibition of splenocyte activation by OP compounds. More importantly, there was essentially no correlation between the pattern of IC50 values for AChE and splenocyte activation. This strongly suggests that acetylcholine and AChE of the type found in the brain are not important in the regulation of splenocyte activation by concanavalin A.

Role of detoxication pathways in acute toxicity levels of phosphorothionate insecticides in the rat.

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.

Target site bioactivation of the neurotoxic organophosphorus insecticide parathion in partially hepatectomized rats.

Target organ bioactivation of phosphorothionate insecticides to their potent anticholinesterase oxon metabolites (for example, parathion to paraoxon) may be extremely important in toxicity because liver and blood provide so much potential protection by a variety of mechanisms, such as the aliesterases which serve as alternate phosphorylation sites. To determine whether the brain can produce sufficient oxon in vivo to contribute to toxicity, male rats were partially hepatectomized and injected i.v. with 1.5 mg/kg parathion. After 30 minutes, brain AChE was inhibited 68% whereas liver and plasma aliesterases were unaffected. Because aliesterases are far more sensitive to paraoxon inhibition than is brain AChE, these results indicate that neither the liver nor extra-hepatic tissues were contributing oxon into the blood stream. Thus target site activation of parathion occurred in vivo at sufficient levels to contribute substantially to toxicity.

Time course of inhibition of acetylcholinesterase and aliesterases following parathion and paraoxon exposures in rats.

The inhibition of cerebral cortex and medulla oblongata acetylcholinesterase and hepatic and plasma aliesterases was studied in female rats following intraperitoneal administration of the phosphorothionate insecticide parathion or its active metabolite paraoxon. Acetylcholinesterase is the target enzyme for organophosphate toxicity while aliesterases are alternative targets for organophosphate inhibition which could serve in a protective capacity during organophosphate intoxication. The effects of pretreatment with cytochrome P450 inducers and inhibitors were also investigated. Pretreatment with phenobarbital slowed the time course of acetylcholinesterase and hepatic aliesterase inhibition following parathion exposure, suggesting the induction of a detoxication pathway(s) to a greater extent than the induction of activation. Although pretreatment with piperonyl butoxide did not affect the rate of acetylcholinesterase inhibition, it slowed hepatic and plasma aliesterase inhibition following parathion administration, presumably from inhibition of the parathion activation pathway. In rats pretreated with beta-naphthoflavone (BNF), hepatic aliesterases demonstrated lower specific activity; additionally, there was a reduced level of inhibition in BNF-pretreated rats following either parathion or paraoxon administration. However, any effects of BNF on parathion- or paraoxon-induced inhibition cannot be distinguished at this time from the effects of the oil vehicle, which reduced esterase inhibition, presumably by its ability to sequester the organophosphate. Brain acetylcholinesterase was partially inhibited before the hepatic aliesterases were maximally inhibited in all treatment groups. In most cases, plasma aliesterases were maximally inhibited within 15 min after organophosphate administration. Thus hepatic aliesterases constitute an important but not completely effective form of protection from the inhibitory effects of organophosphates.

Activation of the phosphorothionate insecticide parathion by rat brain in situ.

The ability of the rat brain to activate the phosphorothionate insecticide parathion to its potent anticholinesterase metabolite paraoxon in situ was observed by ligating the posterior portion of the circulatory system and thus removing the liver from the circulation. Under these conditions no acetylcholinesterase inhibition was observed in 15 min at a dosage of parathion (nominally 2.4 mg/kg) which yielded 95% inhibition when the liver was in the circulation. However, at a higher dose (nominally 48 mg/kg) there was substantial (about 70%) inhibition of brain acetylcholinesterase after 15 min, suggesting that the brain does have the ability to activate parathion in the intact situation.

Short-term effects of paraoxon and atropine on schedule-controlled behavior in rats.

The effects of lethal (2.0 mg/kg) and high sublethal (1.3 mg/kg) dosages of the organophosphate acetylcholinesterase (AChE) inhibitor paraoxon on FR10 performance rate was determined 1 and 2 days after intoxication. The lethal doses were antidoted with either centrally acting atropine sulfate (AS), or atropine methyl bromide (AMB) or atropine methyl nitrate (AMN), both quaternary salts and not expected to act centrally. AChE inhibition in the brain was about 35-60% on the second day after treatment. AS yielded a small transient depression in performance, while AMB and AMN yielded severe deficits, with incomplete recovery. Performance was depressed by 1.3 mg/kg paraoxon by 52% and 34% on days 1 and 2, respectively, while performance was more greatly depressed by the lethal dose, especially with the noncentrally acting antidotes: AS, 67 and 48%; AMB, 81 and 55%; AMN, 91 and 78%. However, a low dose of AS with 2 mg/kg paraoxon resulted in very severe, nonrecovering deficits. A lethal dose of the nonpersistent anti-AChE eserine sulfate, antidoted with a low dose of AS, yielded no deficits. Thus, a high level, acute intoxication with paraoxon yields behavioral deficits which are attenuated by high levels of a centrally acting muscarinic receptor antagonist. The paraoxon-induced performance deficits or their recovery do not correlate directly with AChE inhibition.

Oxidative desulfuration of chlorpyrifos, chlorpyrifos-methyl, and leptophos by rat brain and liver.

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.

Potential immunomodulatory activity of phenylphosphonothioates.

In the course of immunotoxicological studies of several organophosphorous compounds, it was noted that some phenylphosphonothioates enhanced the activation of rat splenocytes by concanavalin A. In the present study some of these compounds were compared to the well-characterized immunomodulatory thiol, 2-mercaptoethanol (2-ME). Two of the compounds are able to substitute for 2-ME and allow near maximal growth of a strictly 2-ME-dependent lymphocyte cell line (CTLL-2). These compounds exhibit essentially additive effects with 2-ME on CTLL-2 cells and rat splenocytes. In agreement with previous results with 2-ME, the phosphonothioates have essentially no effect on interleukin-2 production in mitogen-stimulated splenocyte cultures. One phosphonothioate, as well as its possible thiol hydrolysis product, supports cell survival and enhanced cystine incorporation by CTLL-2 cells. However, 2-ME was more effective in stimulation of enhanced generation of extracellular thiols than were the other agents. These results suggest very similar modes of action for phosphonothionates and 2-ME. Finally, a phosphonothioate selected for low neurotoxic potential was examined and found to effectively support lymphocyte growth in vitro and to exhibit relatively low acute toxicity in mice.