Early differential cell death and survival mechanisms initiate and contribute to the development of OPIDN: a study of molecular, cellular, and anatomical parameters.

Organophosphorus-ester induced delayed neurotoxicity (OPIDN) is a neurodegenerative disorder characterized by ataxia progressing to paralysis with a concomitant central and peripheral, distal axonapathy. Diisopropylphosphorofluoridate (DFP) produces OPIDN in the chicken that results in mild ataxia in 7-14 days and severe paralysis as the disease progresses with a single dose. White leghorn layer hens were treated with DFP (1.7 mg/kg, sc) after prophylactic treatment with atropine (1mg/kg, sc) in normal saline and eserine (1mg/kg, sc) in dimethyl sulfoxide. Control groups were treated with vehicle propylene glycol (0.1 ml/kg, sc), atropine in normal saline and eserine in dimethyl sulfoxide. The hens were euthanized at different time points such as 1, 2, 5, 10 and 20 days, and the tissues from cerebrum, midbrain, cerebellum, brainstem and spinal cord were quickly dissected and frozen for mRNA (northern) studies. Northern blots were probed with BCL2, GADD45, beta actin, and 28S RNA to investigate their expression pattern. Another set of hens was treated for a series of time points and perfused with phosphate buffered saline and fixative for histological studies. Various staining protocols such as Hematoxylin and Eosin (H&E); Sevier-Munger; Cresyl echt Violet for Nissl substance; and Gallocynin stain for Nissl granules were used to assess various patterns of cell death and degenerative changes. Complex cell death mechanisms may be involved in the neuronal and axonal degeneration. These data indicate altered and differential mRNA expressions of BCL2 (anti apoptotic gene) and GADD45 (DNA damage inducible gene) in various tissues. Increased cell death and other degenerative changes noted in the susceptible regions (spinal cord and cerebellum) than the resistant region (cerebrum), may indicate complex molecular pathways via altered BCL2 and GADD45 gene expression, causing the homeostatic imbalance between cell survival and cell death mechanisms. Semi quantitative analysis revealed that the order of severity of damage declines from the spino-cerebellar, ventral, and dorsal tract respectively, suggesting neuroanatomical specificity. Thus, early activation of cell death and cell survival processes may play significant role in the clinical progression and syndromic clinical feature presentation of OPIDN.

In vitro metabolism and interactions of pyridostigmine bromide, N,N-diethyl-m-toluamide, and permethrin in human plasma and liver microsomal enzymes.

1. The in vitro human plasma activity and liver microsomal metabolism of pyridostigmine bromide (PB), a prophylactic treatment against organophosphate nerve agent attack, N,N-diethyl-m-toluamide (DEET), an insect repellent, and permethrin, a pyrethroid insecticide, either alone or in combination were investigated. 2. The three chemicals disappeared from plasma in the following order: permethrin > PB > DEET. The combined incubation of DEET with either permethrin or PB had no effect on permethrin or PB. Binary incubation with permethrin decreased the metabolism of PB and its disappearance from plasma and binary incubation with PB decreased the metabolism of permethrin and its clearance from plasma. Incubation with PB and/or permethrin shortened the DEET terminal half-life in plasma. These agents behaved similarly when studied in liver microsomal assays. The combined incubation of DEET with PB or permethrin (alone or in combination) diminished DEET metabolism in microsomal systems. 3. The present study evidences that PB and permethrin are metabolized by both human plasma and liver microsomal enzymes and that DEET is mainly metabolized by liver oxidase enzymes. Combined exposure to test chemicals increases their neurotoxicity by impeding the body's ability to eliminate them because of the competition for detoxifying enzymes.

Sarin: health effects, metabolism, and methods of analysis.

Sarin (O-isopropylmethylphosphonofluoridate) is a highly toxic nerve agent produced for chemical warfare. Sarin is an extremely potent acetylcholinesterase (AchE) inhibitor with high specificity and affinity for the enzyme. Death by sarin is due to anoxia resulting from airway obstruction, weakness of the muscles of respiration, convulsions and respiratory failure. The main clinical symptoms of acute toxicity of sarin are seizures, tremors and hypothermia. Exposure to sarin during incidents in Japan in 1994, 1995 and 1998, and possible exposure to low levels of sarin during the Gulf War, resulted in the deaths and injury of many people in Japan and caused possible long-term health effects on Gulf War veterans. Symptoms related to sarin poisoning in Japan still exist 1-3 years after the incident and include fatigue, asthenia, shoulder stiffness and blurred vision. Sarin produced seizures in rats and pigs. Recent studies showed that long-term exposure to low levels of sarin caused neurophysiological and behavioral alterations. Toxicity from sarin significantly increased following concurrent exposure to other chemicals such as pyridostigmine bromide. Further research to examine effects of sarin on the cellular and the molecular levels, gene transcription, endocrine system as well as its long-term impact is needed.

Acute exposure to sarin increases blood brain barrier permeability and induces neuropathological changes in the rat brain: dose-response relationships.

We hypothesize that a single exposure to an LD(50) dose of sarin induces widespread early neuropathological changes in the adult brain. In this study, we evaluated the early changes in the adult brain after a single exposure to different doses of sarin. Adult male rats were exposed to sarin by a single intramuscular injection at doses of 1, 0.5, 0.1 and 0.01 x LD(50). Twenty-four hours after the treatment, both sarin-treated and vehicle-treated (controls) animals were analyzed for: (i) plasma butyrylcholinesterase (BChE) activity; (ii) brain acetylcholinesterase (AChE) activity, (iii) m2 muscarinic acetylcholine receptor (m2 mAChR) ligand binding; (iv) blood brain barrier (BBB) permeability using [H(3)]hexamethonium iodide uptake assay and immunostaining for endothelial barrier antigen (EBA); and (v) histopathological changes in the brain using H&E staining, and microtubule-associated protein (MAP-2) and glial fibrillary acidic protein immunostaining. In animals treated with 1 x LD(50) sarin, the significant changes include a decreased plasma BChE, a decreased AChE in the cerebrum, brainstem, midbrain and the cerebellum, a decreased m2 mAChR ligand binding in the cerebrum, an increased BBB permeability in the cerebrum, brainstem, midbrain and the cerebellum associated with a decreased EBA expression, a diffuse neuronal cell death and a decreased MAP-2 expression in the cerebral cortex and the hippocampus, and degeneration of Purkinje neurons in the cerebellum. Animals treated with 0.5 x LD(50) sarin however exhibited only a few alterations, which include decreased plasma BChE, an increased BBB permeability in the midbrain and the brain stem but without a decrease in EBA expression, and degeneration of Purkinje neurons in the cerebellum. In contrast, animals treated with 0.1 and 0.01 x LD(50) did not exhibit any of the above changes. However, m2 mAChR ligand binding in the brainstem was increased after exposure to all doses of the sarin.Collectively, the above results indicate that, the early brain damage after acute exposure to sarin is clearly dose-dependent, and that exposure to 1 x LD(50) sarin induces detrimental changes in many regions of the adult rat brain as early as 24 hours after the exposure. The early neuropathological changes observed after a single dose of 1 x LD(50) sarin could lead to a profound long-term neurodegenerative changes in many regions of the brain, and resulting behavioral abnormalities.

A solid phase extraction reversed-phase HPLC method for the simultaneous determination of methoprene, permethrin and selected metabolites in rat plasma and urine.

A method was validated and applied for the analysis of the insect growth regulator methoprene [Isopropyl (2E,4E)-11-methoxy-3,7,11-trimethyldodeca-2,4-dienoate], its metabolite methoprene acid, the insecticide permethrin [3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester], and two of its metabolites, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, in rat plasma and urine using solid-phase extraction and reversed-phase high performance liquid chromatography. The analytes were separated using gradient of 55-100% acetonitrile in water (pH 4.0) at a flow rate ranging between 0.6 and 1.0 mL/min over a period of 20 min, and UV detection at 210 and 254 nm. The retention times ranged from 7.3 to 18.4 min. The limits of detection ranged between 50 and 100 ng/ml, while limits of quantitation were 100-150 ng/mL. Average percentage recovery of five spiked plasma samples was 83.6 +/- 3.9, 80.1 +/- 5.4, 82.1 +/- 4.4, 83.7 +/- 3.9 and 83.1 +/- 4.7, and from urine 79.3 +/- 4.3, 82.0 +/- 5.4, 80.7 +/- 4.2, 78.9 +/- 5.7 and 83.9 +/- 4.5 for methoprene, methoprene acid, permethrin, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The method was linear and reproducible over the range of 100-1000 ng/mL. This method was applied to analyze the above chemicals and metabolites following their combined administration in rats.

DEET (N,N-diethyl-m-toluamide) alone and in combination with permethrin increased urinary excretion of 6beta-hydroxycortisol in rats, a marker of hepatic CYP3A induction.

In this study, the ratio of 6beta-hydroxycortisol (6beta-OHF) to free cortisol (F) was determined in urine following a single dermal dose of 400 mg/kg of DEET (N,N-diethyl-m-toluamide), and 1.3 mg/kg of permethrin, alone and in combination, in rats. Urine samples were collected at 2, 4, 8, 16, 24, 48, and 72 h after application. Recoveries of 6beta-OHF and cortisol (F) from control urine samples were between 75 and 85%, with limits of detection at 30 and 10 ng/ml for cortisol and 6beta-OHF, respectively. A single dermal dose of DEET alone and in combination with permethrin significantly increased urinary excretion of 6beta-hydroxycortisol 24 h after dosing. Permethrin did not significantly alter the urinary excretion of 6beta-hydroxycortisol. These results indicate that DEET, alone and in combination with permethrin, increased urinary excretion of 6beta-OHF in rats following a single dermal dose application.

Absorption, distribution, metabolism and excretion of daily oral doses of [14C]methyl parathion in hens.

Adult hens were given oral daily doses of 2 mg (2 microC(i))/kg/day (14% of oral LD(50) in male rats) of [14C]methyl parathion (O,O-dimethyl O-4-nitrophenyl phosphorothioate) for 10 consecutive days. Five treated hens were sacrificed at 1, 2, 4, 8, 12, 24, and 48 h after the last dose. Methyl parathion was absorbed from the gastrointestinal tract and distributed rapidly. Maximum radioactivity was detected in tissues within 8 h of dosing, (ng methyl parathion equivalent/g fresh tissue or ml plasma): Plasma (189.2), liver (94.7), kidney (146.2), brain (61.4), gastrointestinal tissues (106.7). Methyl parathion was detected in the plasma, kidney and liver, while methyl parathion metabolite p-nitrophenol was detected in the liver and in the kidney. Elimination of methyl parathion from plasma was monophasic with a terminal half-life of 17.5 h, corresponding to an elimination rate constant of 0.039 ng/hr. Most of the absorbed radioactivity was excreted in the combined fecal-urine excreta (98%). Analysis of the metabolites in the excreta revealed that non-conjugated metabolites accounted for 13% of the total excretion. Conjugated metabolites accounted for 87% of the total excretion; of that, 6% as p-nitrophenyl-glucoronide conjugate, 7% as p-nitrophenyl-sulfate conjugate, 23% as bound hot sulfuric acid hydrolyzable residues, and 51% as water soluble metabolites. The presence of majority of radioactivity in the excreta as conjugated metabolites indicates that determining only unbound p-nitrophenol as a biological marker for methyl parathion exposure underestimates total fecal-urine excretion of p-nitrophenol. The slow elimination rate of methyl parathion is significant, since hens are more comparable to humans with respect to their cytochrome P450 activities.

A validated HPLC method for the determination of pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine in rat plasma and urine.

A method was developed for the separation and quantification of the anti-nerve agent pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), the analgesic drugs acetaminophen and acetylsalicylic acid, and the stimulant caffeine (3,7-dihydro-1,3,7-trimethyl-1-H-purine-2,6-dione) in rat plasma and urine. The compounds were extracted using C(18) Sep-Pak(R) cartridges then analyzed by high performance liquid chromatography (HPLC) with reversed phase C18 column, and UV detection at 280 nm. The compounds were separated using gradient of 1-85% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.5 ml/min in a period of 14 min. The retention times ranged from 8.8 to 11.5 min. The limits of detection were ranged between 100 and 200 ng/ml, while limits of quantitation were 150-200 ng/ml. Average percentage recovery of five spiked plasma samples were 70.9+/-9.5, 73.7+/-9.8, 88.6+/-9.3, 83.9+/-7.8, and from urine 69.1+/-8.5, 74.5+/-8.7, 85.9+/-9.8, 83.2+/-9.3, for pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine, respectively. The relationship between peak areas and concentration was linear over range between 100 and 1000 ng/ml. The resulting chromatograms showed no interfering peaks from endogenous plasma or urine components. This method was applied to analyze these compounds following oral administration in rats.

Subchronic dermal application of N,N-diethyl m-toluamide (DEET) and permethrin to adult rats, alone or in combination, causes diffuse neuronal cell death and cytoskeletal abnormalities in the cerebral cortex and the hippocampus, and Purkinje neuron loss in the cerebellum.

N,N-Diethyl m-toluamide (DEET) and permethrin have been implicated as potential neurotoxic agents that may have played an important role in the development of illnesses in some veterans of the Persian Gulf War. To determine the effect of subchronic dermal application of these chemicals on the adult brain, we evaluated histopathological alterations in the brain of adult male rats following a daily dermal dose of DEET (40 mg/kg in 70% ethanol) or permethrin (0.13 mg/kg in 70% ethanol) or a combination of the two for 60 days. Control rats received a daily dermal dose of 70% ethanol for 60 days. Animals were perfused and brains were processed for morphological and histopathological analyses following the above regimen. Quantification of the density of healthy (or surviving) neurons in the motor cerebral cortex, the dentate gyrus, the CA1 and CA3 subfields of the hippocampus, and the cerebellum revealed significant reductions in all three treated groups compared with the control group. Further, animals receiving either DEET or permethrin exhibited a significant number of degenerating (eosinophilic) neurons in the above brain regions. However, degenerating neurons were infrequent in animals receiving both DEET and permethrin, suggesting that neuronal cell death occurs earlier in animals receiving combined DEET and permethrin than in animals receiving either DEET or permethrin alone. The extent of neuron loss in different brain regions was similar among the three treatment groups except the dentate gyrus, where neurodegeneration was significantly greater with exposure to DEET alone. The neuron loss in the motor cerebral cortex and the CA1 subfield of all treated groups was also corroborated by a significant decrease in microtubule associated protein 2-immunoreactive elements (15-52% reduction), with maximal reductions occurring in rats receiving DEET alone; further, the surviving neurons in animals receiving both DEET and permethrin exhibited wavy and beaded dendrites. Analysis of glial fibrillary acidic protein immunoreactivity revealed significant hypertrophy of astrocytes in the hippocampus and the cerebellum of all treated groups (24-106% increase). Thus, subchronic dermal application of DEET and permethrin to adult rats, alone or in combination, leads to a diffuse neuronal cell death in the cerebral cortex, the hippocampal formation, and the cerebellum. Collectively, the above alterations can lead to many physiological, pharmacological, and behavioral abnormalities, particularly motor deficits and learning and memory dysfunction.

High performance liquid chromatographic determination of diazinon, permethrin, DEET (N, N-diethyl-m-toluamide), and their metabolites in rat plasma and urine.

A rapid method was developed for the analysis of the insecticide (A) diazinon (O,O-diethyl O-2-isopropyl-6-methylpyridimidinyl) phosphorothioate, its metabolites (B) diazoxon (O,O-diethyl O-2-isopropyl-6-methylpyridimidinyl) phosphate, and (C) 2-isopropyl-6-methyl-4-pyrimidinol, the insecticide (D) permethrin [3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid (3-phenoxyphenyl)methylester], its metabolites (E) m-phenoxybenzyl alcohol, and (F) m-phenoxybenzoic acid, the insect repellent (G) DEET (N,N-diethyl-m-toluamide), and its metabolites (H) m-toluamide and (I) m-toluic acid in rat plasma and urine. The method is based on using C18 Sep-Pak cartridges (Waters Corporation, Milford, Mass., U.S.A.) for solid phase extraction and high performance liquid chromatography with a reversed phase C18 column, and absorbance detection at 230 nm for compounds A, B, and C, and at 210 nm for compounds D-I. The compounds were separated using a gradient from 1% to 99% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.7 mL/min in a period of 17 min. The limits of detection were ranged between 20 and 100 ng/mL, while limits of quantification were 80-200 ng/mL. The relationship between peak areas and concentration was linear over a range of 100-1000 ng/mL. This method was applied to determine the above insecticides and their metabolites following dermal administration in rats.