Janice E. Chambers

Activation and degradation of the phosphorothionate insecticides parathion and EPN by rat brain

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.

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.

Inhibition Patterns of Brain Acetylcholinesterase and Hepatic and Plasma Aliesterases Following Exposures to Three Phosphorothionate Insecticides and Their Oxons in Rats

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.

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.

Biochemical mechanisms contributing to species differences in insecticidal toxicity

Comparison of published LD50 or LC50 levels for a variety of insecticides in several vertebrate species indicate that a wide range of toxicity levels exist, and these cannot be easily predicted within either a chemical group or within a species. There is a relatively limited data base documenting interactions between insecticides and other chemicals, either agricultural or non-agricultural; however, the fact that all major insecticide groups perturb nervous system function as their primary mechanism of acute toxicity suggests the potential for interactions. Studies in our laboratories on a select group of phosphorothionate insecticides in rats indicated that brain acetylcholinesterase sensitivity to inhibition by the oxons, the active metabolites of the phosphorothionates, does not correlate with acute toxicity levels. The activities and properties of hepatic cytochrome P450-mediated activation (desulfuration) and detoxication (dearylation) of the phosphorothionates as well as of A-esterase-mediated hydrolysis of oxons contribute substantially to understanding the acute toxicity levels in rats, as does the sensitivity of the protective aliesterases to phosphorylation. However, in the channel catfish, the acetylcholinesterase sensitivity to oxon inhibition reflects the acute toxicity level of these same insecticides, and may be largely responsible for determining the acute toxicity level in this species. Thus, metabolism of insecticides appears to be far more influential in some species than others in determining the toxicity elicited.

The Acquisition and Application of Absorption, Distribution, Metabolism, and Excretion (ADME) Data in Agricultural Chemical Safety Assessments

A proposal has been developed by the Agricultural Chemical Safety Assessment (ACSA) Technical Committee of the ILSI Health and Environmental Sciences Institute (HESI) for an improved approach to assessing the safety of crop protection chemicals. The goal is to ensure that studies are scientifically appropriate and necessary without being redundant, and that tests emphasize toxicological endpoints and exposure durations that are relevant for risk assessment. Incorporation of pharmacokinetic studies describing absorption, distribution, metabolism, and excretion is an essential tool for improving the design and interpretation of toxicity studies and their application for safety assessment. A tiered approach is described in which basic pharmacokinetic studies, similar to those for pharmaceuticals, are conducted for regulatory submission. Subsequent tiers provide additional information in an iterative manner, depending on pharmacokinetic properties, toxicity study results, and the intended uses of the compound.

Xenobiotic biotransformation systems in fishes

Abstract The early speculations that fishes would not possess detoxication sytems because of the excretory activity of the gill have been shown to be wrong. Indeed, fishes are capable of xenobiotic metabolism by both microsomal oxidation, reduction and conjugation. Enzyme characteristics appear to be similar between fishes and mammals, with the exception of typically lower temperature optima in fishes which may reflect the usual lower operating temperatures of heterotherms as compared to homeotherms. Early reports of litte or no enzyme activity in fishes may very well have reflected a lack of knowledge of the lower temperature optima in fishes. However, even under optimized conditions, fishes demonstrate lower enzyme activities than mammals. Although reports are fragmentary, evolutionary trends can be observed among fishes with some biotransformation systems absent or less developed in the more primitive fishes as compared with more advanced fishes. The less effective induction system in fishes as compared to mammals might reflect the fact that fishes live in an environment in which toxicants are diluted, thereby reducing the response of enzymes to a xenobiotic challenge. The real significance of the xenobiotic biotransformation enzymes to the fish from an in vivo standpoint has not been extensively investigated. In a few cases the metabolism of a xenobiotic by fishes is known to affect its toxicity and it is entirely possible that the lower enzyme activities that fishes possess are sufficient for the disposition of the concentrations of toxicants that fishes ordinarily encounter. However, the aquatic environment is becoming more contaminated than ever before. From the little information available, we cannot predict whether fishes have the capability to adapt to and process these higher concentrations of toxicants. Therefore, further study of the biotransformation systems is essential so that an evaluation of fish-toxicant interactions can be made.

Inhibition of acetylcholinesterase and aliesterases of fingerling channel catfish by chlorpyrifos, parathion, and S,S,S-tributyl phosphorotrithioate (DEF)

Abstract Serine esterases can be inhibited by organophosphorus compounds. The in vitro potency of the organophosphorus pesticide DEF and oxons of the phosphorothionate insecticides chlorpyrifos (chlorpyrifos-oxon, Cpxn) and parathion (paraoxon, Pxn) were determined for brain, gill, liver, muscle, and plasma for acetylcholinesterase (AChE) and gill, liver, and plasma aliesterases (ALiE). AChE activity was inhibited less than 15% in all tissues in the presence of 1 mM DEF®. AChE I50 values for Cpxn were 28–33 nM, and for Pxn were 446–578 nM. ALiE I50 values for Cpxn were 0.1–0.2 nM, for DEF were 24–163 nM, and for Pxn were 6–46 nM. Fish were exposed to chlorpyrifos (Cp), parathion (Pth), DEF, and combinations of the phosphorothionates with DEF for 4 h followed by a 384-h recovery period. AChE inhibition following Cp and Pth exposures was rapid. Cp led to more persistent inhibition than Pth. DEF treatments yielded low levels of AChE inhibition in brain and muscle, and high levels of inhibition in gill, liver, and plasma. In vitro and in vivo results suggest that DEFs disposition and/or mode of action are different than those of Cp or Pth. Exposure to DEF resulted in persistent, high level inhibition of ALiE activity. Greater AChE inhibition in DEF plus Cp or Pth treatments was not evident, suggesting that ALiEs do not serve to appreciably protect AChE in channel catfish, even though ALiEs are inherently more sensitive to inhibition.

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.