Edward C. Meek

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.

Testing of novel brain-penetrating oxime reactivators of acetylcholinesterase inhibited by nerve agent surrogates.

A critical need for combating the effects of organophosphate (OP) anticholinesterases, such as nerve agents, is the current lack of an effective oxime reactivator which can penetrate the blood-brain barrier (BBB), and therefore reactivate inhibited acetylcholinesterase (AChE) in the brain. Our laboratories have synthesized and have initiated testing of novel phenoxyalkyl pyridinium oximes (patent pending) that are more lipophilic than currently approved oximes. This is a preliminary report on these novel oximes which have been tested in vitro in rat brain homogenates with highly relevant surrogates for sarin (phthalimidyl isopropyl methylphosphonate; PIMP) and VX (nitrophenyl ethyl methylphosphonate; NEMP). The oximes demonstrated a range of 14-76% reactivation of rat brain AChE in vitro. An in vivo testing paradigm was developed in which the novel oxime was administered at the time of maximal brain AChE inhibition (about 80%) (1h) elicited by nitrophenyl isopropyl methylphosphonate (NIMP; sarin surrogate). This paradigm, with delayed administration of oxime to a time when brain AChE was starting to recover, was designed to minimize reactivation/reinhibition of peripheral AChE during the reactivation period which would decrease the availability of the surrogate for entry into the brain; this paradigm will allow proof of concept of BBB penetrability. The initial studies of these oximes in vivo with the sarin surrogate NIMP have indicated reactivation of up to about 25% at 30 min after oxime administration and substantial attenuation of seizure behavior from some of the oximes. Therefore these novel oximes have considerable potential as brain-protecting therapeutics for anticholinesterases.

Exposure to p,p'-dichlorodiphenyldichloroethylene (DDE) induces fasting hyperglycemia without insulin resistance in male C57BL/6H mice.

Approximately 8.3% of the United States (U.S.) population have either diagnosed or undiagnosed diabetes mellitus. Out of all the cases of diabetes mellitus, approximately 90-95% of these cases are type 2 diabetes mellitus (T2D). Although the exact cause of T2D remains elusive, predisposing factors include age, weight, poor diet, and a sedentary lifestyle. Until recently the association between exposure to environmental contaminants and the occurrence of diabetes had been unexplored. However, recent epidemiological studies have revealed that elevated serum concentrations of certain persistent organic pollutants (POPs), especially organochlorine pesticides, are positively associated with increased prevalence of T2D and insulin resistance. The current study seeks to investigate if this association is causative or coincidental. Male C57BL/6H mice were exposed to DDE (2.0mg/kg or 0.4mg/kg) or vehicle (corn oil; 1mL/kg) for 5 days via oral gavage; fasting blood glucose, glucose tolerance, and insulin challenge tests were performed following a 7 day resting period. Exposure to DDE caused significant hyperglycemia compared to vehicle and this hyperglycemic effect persisted for up to 21 days following cessation of DDE administration. Intraperitoneal glucose tolerance tests and phosphorylation of Akt in the liver, skeletal muscle, and adipose tissue following insulin challenge were comparable between vehicle and DDE treated animals. To determine the direct effect of exposure to DDE on glucose uptake, in vitro glucose uptake assays following DDE exposure were performed in L6 myotubules and 3T3-L1 adipocytes. In summary, subacute exposure to DDE does produce fasting hyperglycemia, but this fasting hyperglycemia does not appear to be mediated by insulin resistance. Thus, the current study reveals that subacute exposure to DDE does alter systemic glucose homeostasis and may be a contributing factor to the development of hyperglycemia associated with diabetes.

Racial Differences in Paraoxonase-1 (PON1): A Factor in the Health of Southerners?

Background The southern United States (excluding Florida) has the highest age-adjusted rate of cardiovascular disease (CVD) in the country, with African Americans having a higher prevalence of CVD than Caucasians. Paraoxonase-1 (PON1), an enzyme associated with high-density lipoprotein particles, participates both in the hydrolysis of oxidized lipids (thus protecting against atherosclerosis) and in the hydrolysis of organophosphates. Higher paraoxonase activity has been associated with lower risk of atherosclerosis. Objectives In this study we characterized the distribution of the functional PON1Q192R polymorphisms (PON status as assessed by diazoxonase to paraoxonase ratios) and the PON1 activity levels in 200 adult males and females of both races (50 in each race/sex class) from the southern United States from commercially obtained blood bank serum samples. Methods We used spectrophotometric methods with serum to determine PON1 status, arylesterase activities (phenyl acetate hydrolysis), and levels of cotinine and C-reactive protein (CRP). Results African Americans had higher paraoxonase activities but lower diazoxonase activities than did Caucasians, consistent with African Americans having a lower proportion of the functional genotype QQ (QQ 15%, QR 34%, RR 44%, 7% indeterminate), than did Caucasians (QQ 60%, QR 31%, RR 7%, 2% indeterminate). Cotinine levels indicated that all samples came from non-smokers and that CRP levels were higher in African Americans than in Caucasians and higher in females than in males. CRP levels showed no association with paraoxonase activities. Conclusions These data present initial observations for use in characterizing the poorer cardiovascular health status of the population in the southern United States and more specifically southern African Americans.

Synthesis and in vitro and in vivo inhibition potencies of highly relevant nerve agent surrogates.

Four nonvolatile nerve agent surrogates, 4-nitrophenyl ethyl dimethylphosphoramidate (NEDPA, a tabun surrogate), 4-nitrophenyl ethyl methylphosphonate (NEMP, a VX surrogate), and two sarin surrogates, phthalimidyl isopropyl methylphosphonate (PIMP) and 4-nitrophenyl isopropyl methylphosphonate (NIMP), were synthesized and tested as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors. These surrogates were designed to phosphorylate cholinesterases with the same moiety as their respective nerve agents, making them highly relevant for the study of cholinesterase reactivators. Surrogates were characterized by liquid chromatography-mass spectrometry and nuclear magnetic resonance. NEMP, PIMP, and NIMP were potent inhibitors of rat brain, skeletal muscle, diaphragm, and serum AChE as well as human erythrocyte AChE and serum BuChE in vitro. PIMP was determined to degrade quickly in aqueous solution, making it useful for in vitro assays only, and NEDPA was not a potent inhibitor of AChE or BuChE in vitro; therefore, these two surrogates were not tested in subsequent in vivo studies. Sublethal dosages (yielding about 80% brain AChE inhibition) were determined for both the stable sarin surrogate, NIMP (0.325 mg/kg ip), and the VX surrogate, NEMP (0.4 mg/kg ip), in adult male rats. Time course studies indicated the time to peak brain AChE inhibition for both NIMP and NEMP to be 1 h postexposure. Both surrogates yielded severe cholinergic signs. These dosages did not require the addition of atropine to prevent lethality, and the rate of AChE aging was slow, making these surrogates useful for reactivation studies both in vitro and in vivo. The surrogates synthesized in this study are potent yet safer to test than nerve agents and are useful tools for initial screening of nerve agent oxime therapeutics.

Novel substituted phenoxyalkyl pyridinium oximes enhance survival and attenuate seizure-like behavior of rats receiving lethal levels of nerve agent surrogates

Novel substituted phenoxyalkyl pyridinium oximes, previously shown to reactivate brain cholinesterase in rats treated with high sublethal dosages of surrogates of sarin and VX, were tested for their ability to prevent mortality from lethal doses of these two surrogates. Rats were treated subcutaneously with 0.6mg/kg nitrophenyl isopropyl methylphosphonate (NIMP; sarin surrogate) or 0.65mg/kg nitrophenyl ethyl methylphosphonate (NEMP; VX surrogate), dosages that were lethal within 24h to all tested rats when they received only 0.65mg/kg atropine at the time of initiation of seizure-like behavior (about 30min). If 146mmol/kg 2-PAM (human equivalent dosage) was also administered, 40% and 33% survival was obtained with NIMP and NEMP, respectively, while the novel Oximes 1 and 20 provided 65% and 55% survival for NIMP and 75 and 65% for NEMP, respectively. In addition, both novel oximes resulted in a highly significant decrease in time to cessation of seizure-like behavior compared to 2-PAM during the first 8h of observation. Brain cholinesterase inhibition was slightly less in novel oxime treated rats compared to 2-PAM in the 24h survivors. The lethality data indicate that 24h survival is improved by two of the novel oximes compared to 2-PAM. The cessation of seizure-like behavior data strongly suggest that these novel oximes are able to penetrate the blood-brain barrier and can combat the hypercholinergic activity that results in seizures. Therefore this oxime platform has exceptional promise as therapy that could both prevent nerve agent-induced lethality and attenuate nerve agent-induced seizures.

Novel Nucleophiles Enhance the Human Serum Paraoxonase 1 (PON1)-mediated Detoxication of Organophosphates

Paraoxonase 1 (PON1) is a calcium-dependent hydrolase associated with serum high-density lipoprotein particles. PON1 hydrolyzes some organophosphates (OPs), including some nerve agents, through nucleophilic attack of hydroxide ion (from water) in the active site. Most OPs are hydrolyzed inefficiently. This project seeks to identify nucleophiles that can enhance PON1-mediated OP 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 submicromolar 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 OPs could be a unique and unprecedented mechanism of antidotal action.

Novel brain‐penetrating oximes for reactivation of cholinesterase inhibited by sarin and VX surrogates

Current oxime reactivators for organophosphate‐inhibited cholinesterase (ChE) do not effectively cross the blood–brain barrier and therefore cannot restore brain ChE activity in vivo. Our laboratories have studied highly relevant sarin and VX surrogates, which differ from their respective nerve agents only in the leaving group and thereby leave ChE phosphylated with the same chemical moiety as sarin and VX. Our laboratories have developed novel substituted phenoxyalkyl pyridinium oximes that lead to reduced ChE inhibition in the brains of rats challenged with a high sublethal dosage of the sarin surrogate, whereas 2‐PAM did not, using a paradigm designed to demonstrate brain penetration. In addition, treatment of rats with these novel oximes is associated with attenuation of seizure‐like behavior compared to rats treated with 2‐PAM, providing additional evidence that the oximes penetrate the blood–brain barrier. Further, some of the oximes provided 24‐h survival superior to 2‐PAM, and shortened the duration of seizure‐like behavior when rats were challenged with lethal dosages of the sarin and VX surrogates, providing additional support for the conclusion that these oximes penetrate the brain.

Human paraoxonase 1 hydrolysis of nanomolar chlorpyrifos-oxon concentrations is unaffected by phenotype or Q192R genotype

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 (320 μM) and low (178 nM) 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±1015 μmol 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±43 nmol 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.

A case-control study: The association of serum paraoxonase 1 activity and concentration with the development of type 2 diabetes mellitus

A longitudinal study assessed serum paraoxonase 1 (PON1) activity and concentration as affected by age and as associated with the development of type 2 diabetes (T2D). PON1s recently established physiological function is the hydrolysis of lipolactones in oxidized LDL particles.