Howard W. Chambers

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.

Noncatalytic detoxication of six organophosphorus compounds by rat liver homogenates

Abstract 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 (n M 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 I 50 s 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.

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.

An investigation of acetylcholinesterase inhibition and aging and choline acetyltransferase activity following a high level acute exposure to paraoxon

Abstract 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.

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.

Biomarkers as Predictors in Health and Ecological Risk Assessment

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.

Chapter 65 – The Metabolism of Organophosphorus Insecticides

Publisher Summary 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.

Chemistry of Organophosphorus Insecticides

Publisher Summary This chapter introduces and summarizes the chemistry of organophosphorus insecticides. A classification scheme has been presented into which all commercially important OP insecticides fit, based on the central phosphorus atom and the four atoms immediately surrounding it. OP insecticides may be considered to be derivatives of phosphoric acid (H3PO4) or phosphonic acid (H3PO3) in which all H atoms are replaced by organic moieties. Thus, phosphates are compounds in which the P atom is surrounded by four O atoms. In many OPs, one or more of the oxygen atoms are replaced by sulfur and/or nitrogen. For phosphoric acid derivatives, the O, S, and N atoms can be arranged in 20 different configurations. Initially, elemental phosphorus is converted into P2S5 by reaction with sulfur or into PCl3 by direct chlorination. Trialklyl phosphites are particularly useful in the preparation of dialkyl vinyl phosphates from α-chloroaldehydes and ketones. For OPs with three different substituents on the P atom, it is necessary to begin with P(:O) Cl3 (from intermediate 3) or P(:S) Cl3 (from intermediate 4). Dialkyl phosphates with phenolic or heterocyclic leaving groups are easily prepared from the phenol or heterocyclic alcohol and the dialkyl phosphorochloridate. Unfortunately, the only phosphorochloridates readily available commercially are the dimethyl and the diethyl, the former being quite unstable. OP insecticides, when kept cool, dark, and anhydrous, are usually quite stable. Exposure to heat, light (especially ultraviolet), and/or water, however, may lead to chemical alterations. The three primary reactions involving the phosphorus atom and those immediately surrounding it are hydrolysis, oxidation, and rearrangement.

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.

Rotenone tolerance in mosquitofish

Abstract Resistant mosquitofish showed a 1·8-fold tolerance to rotenone over a susceptible strain. The 24-h LC 50 values for rotenone in susceptible and resistant mosquitofish were 0·017 ppm and 0·031 ppm, respectively. Results with sesamex, an inhibitor of mixed-function oxidase (mfo) enzymes, indicated that rotenone tolerance in mosquitofish is solely the result of increased levels of mfo enzymes.