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.

Delayed neurotoxicity of phenylphosphonothioate esters.

Administration of a single oral dose of five phenylphosphonothioate esters produced delayed neurotoxicity in hens; their potency was, in descending order, cyanofenphos, EPN, desbromoleptophos, leptophos, and EPBP (Seven). Histological examination showed that in some hens there was marked axonal and myelin degeneration in the spinal cord and peripheral nerves. The results suggest that delayed neurotoxicity may be a general feature of phenylphosphonothioate insecticides.

Tobacco budworm P-glycoprotein: biochemical characterization and its involvement in pesticide resistance.

Since pesticides have been shown to interact with P-glycoprotein (P-gp), the purpose of this study was to examine the possible role of P-gp in pesticide resistance in the tobacco budworm (Heliothis virescens). Using three P-gp antibodies, P-gp expression in various resistant populations of tobacco budworms was found to be 2-6-times that of the susceptible larvae. Tobacco budworm P-gp was glycosylated and localized primarily in the cuticle and fat body with little expression in the mid gut. To determine the role of P-gp in pesticide resistance, resistant tobacco budworm larvae were treated with a P-gp inhibitor, quinidine, and challenged with various doses of thiodicarb. Inhibition of P-gp decreased the LD50 for thiodicarb by a factor of 12.5. Quinidine treatment did not result in a significant inhibition of the P-450 system nor did it alter the feeding of the larvae, suggesting the potential involvement of P-gp in pesticide resistance. An age-dependent increase in P-gp expression was detected in resistant larvae as compared to control, susceptible larvae. This correlates with the reported age-dependent increase in resistance and is further evidence supporting the role of P-gp in the development of pesticide resistance.

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.

The organophosphate pesticide chlorpyrifos affects form deprivation myopia.

PURPOSE: The effects of the anti-cholinesterase organophosphate pesticide chlorpyrifos (CPF) on the refractive development of the eye were examined. Form deprivation was used to induce eye growth to address the previously reported relationship between organophosphate pesticide use and the incidence of myopia. METHODS: Chickens, a well-established animal model for experimental myopia and organophosphate neurotoxicity, were dosed with chlorpyrifos (3 mg/kg per day, orally, from day 2 to day 9 after hatching) or corn oil vehicle (VEH) with or without monocular form deprivation (MFD) over the same period. The set of dependent measures included the refractive state of each eye measured using retinoscopy, axial dimensions determined with A-scan ultrasound, and intraocular pressure. RESULTS: Dosing with CPF yielded an inhibition of 35% butyrylcholinesterase in plasma and 45% acetylcholinesterase in brain. MFD resulted in a significant degree of myopia in form-deprived eyes resulting from significant lengthening of the vitreal chamber of the eye. CPF significantly reduced the effect of MFD, resulting in less myopic eyes (mean refraction: VEH-MFD = -16.2 +/- 2.3 diopters; CPF-MFD = -11.1 +/- 1.8 diopters) with significantly shorter vitreal chambers. Nonoccluded eyes were, on average, slightly hyperopic. Treatment with CPF for 1 week in the absence of MFD led to no significant change in ocular dimensions or refraction relative to controls. CONCLUSIONS: The use of form deprivation as a challenge suggests that CPF treatment interferes with the visual regulation of eye growth.

Persistent alterations of calmodulin kinase II activity in chickens after an oral dose of tri-o-cresyl phosphate.

Calmodulin kinase II has been found to be involved in the increased phosphorylation of brain microtubule and spinal cord neurofilament triplet proteins following treatment of animals with organophosphorus compounds that are capable of producing organophosphorus compound-induced delayed neurotoxicity (OPIDN). In this report, chickens were given a single oral neurotoxic dose of 750 mg/kg tri-o-cresyl phosphate (TOCP), and killed after 1 or 21 days of treatment. Crude calmodulin kinase II from brain cytosol as well as phosphocellulose-purified microtubules were prepared from control and treated animals. Phosphorylation reactions were started by adding protein into the phosphorylation buffer in the presence of Mg2+, Ca2+, calmodulin or trifluoperazine, and [gamma-32P]ATP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to autoradiography. The extent of the calmodulin kinase II autophosphorylation as well as the Ca2+/calmodulin-dependent phosphorylation of the purified microtubules was investigated. The enzyme activities isolated from control and treated animals were compared. Autophosphorylation of calmodulin kinase II was found to be higher in both 1-day and 21-day TOCP-treated animals than in control animals. The activity of the kinase to phosphorylate exogenous substrates such as tubulin and microtubule-associated protein-2 (MAP-2) was also higher in the treated hens than in the controls. The increased activity of the kinase was noted at day 1 following treatment when no clinical signs were observed and persisted until day 21 when the animals were paralyzed completely. This finding supports the significance of altered calmodulin kinase II in the pathogenesis of OPIDN.

Ca2+/calmodulin-dependent protein kinase II from hen brain. Purification and characterization.

Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) has been purified from hen whole brain. The enzyme was purified 3000-fold using phosphocellulose and calmodulin-Agarose column chromatography. The specific activity was 200 nmol/min/mg protein. Microtubule associated protein-2 (MAP-2) was used as a substrate to assess the activity of the enzyme during purification and for its characterization. CaM-kinase II consisted of alpha and beta/beta' subunits of molecular weights 46,000 and 55,000/52,000, respectively. The ratio of alpha to beta/beta' subunits was 3:1 in the enzyme purified from the whole brain. The enzyme exhibited broad substrate specificity and phosphorylated myelin basic protein, MAP-2, histone II, histone VIII, casein, tubulin, myosin light chains, glycogen synthase, and phosvitin in decreasing order. Phosphorylase b was phosphorylated at a negligible rate. Autophosphorylation of CaM-kinase II for 10 min in the presence of calcium and calmodulin decreased its total activity to 33%, and calcium/calmodulin-independent activity reached 30% after 1 min and then dropped to 14% after 10 min of autophosphorylation. The Km value of ATP was 19 +/- 1.3 microM, and the K0.5 values of calcium and calmodulin were 4.4 +/- 0.5 and 3.0 +/- 0.5 microM, respectively. The latter were determined using myelin basic protein as the substrate. CaM-kinase II exhibited great differences in the calmodulin requirement for phosphorylation of MAP-2, histone II and myelin basic protein. MAP-2 required the least amount of calmodulin for its phosphorylation. Autophosphorylation of CaM-kinase II resulted in decreased mobility of the alpha-subunit but apparently not of the beta/beta' subunits in sodium dodecyl/sulfate-polyacrylamide gel. Antiserum was raised against the CaM-kinase II alpha subunit and used for testing cross-reactivity of hen brain enzyme with that of other species. The antiserum which reacted with both alpha and beta subunits of hen brain CaM-kinase II cross-reacted with only the alpha subunit of rat, mouse, rabbit, cat, dog, pig and human brain samples. The purified hen brain CaM-kinase II is a multifunctional enzyme and resembled rat brain CaM-kinase II in several properties. Immunocross-reactivity suggested that there was similarity in the alpha but not the beta/beta' subunits of the hen brain enzyme and the brain enzyme of other species.

Enhanced mRNA expression of neurofilament subunits in the brain and spinal cord of diisopropyl phosphorofluoridate-treated hens.

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester, and a single injection of this compound (1.7 mg/kg, s.c.) produces delayed neurotoxicity (OPIDN) in hens in 7-14 days. Clinically, the disease is marked by hindlimb ataxia followed by paralysis after some time. A characteristic feature of this neuropathy is axonal swelling in the initial stages and comparative dissolution of the accumulated material and degeneration of distal axons with disease progression. Axonal swelling consists of aggregated neurofilaments, microtubules, and proliferated smooth endoplasmic reticulum. We studied expression of neurofilament (NF) mRNAs in brain regions and spinal cord to elucidate their role in OPIDN. There was a 50-200% increase in NF transcripts in 24 hr after DFP administration. The NF-L mRNA level started falling after 1-5 days and came down to control level in susceptible brain regions (i.e. cerebellum and brainstem) and spinal cord, but not in cerebral cortex, which does not show degeneration of axons in OPIDN. Cerebral cortex exhibited elevated levels of both NF-L and NF-M transcripts in DFP-treated hens throughout the period of observation. The induction of NF messages is consistent with the previously reported effect on extension of neurites of human neuroblastoma cells in culture. The transient increase in NF messages in susceptible tissues either may be responsible for the delayed degeneration of axons in OPIDN or is the result of interruption of regulatory signal due to progressive degeneration of axons.

Alteration in neurofilament axonal transport in the sciatic nerve of the diisopropyl phosphorofluoridate (DFP)-treated hen.

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester that produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in hens 7-14 days after a single s.c. dose of 1.7 mg/kg. In this study, hens were treated with a single dose of DFP (1.7 mg/kg, s.c.) 24 hr after [35S]methionine injection into the sacrolumbar region of their spinal cord, and killed 3, 7, 14, or 27 days post-DFP treatment. The rates of transport of labeled high (NF-H), medium (NF-M), and low (NF-L) molecular weight neurofilaments, and tubulin were faster in DFP-treated birds than in controls after 3 days. Subsequently, the rate of transport of these proteins started falling, so that the peaks of labeled proteins in control and DFP-treated hens were overlapping after 7 days. At 14 days, the peaks of NF-H, NF-M, and NF-L in treated hens were distinctly behind the corresponding peaks in control hens. This was again followed by an increase in transport of NF-H and NF-L, but not of NF-M, so that the labeled NF-H and NF-L showed the same pattern in control and treated hens after 27 days. The transient decrease in NF-H and NF-L axonal transport rate, and recovery correlated in a temporal manner with the previously reported increase of Ca2+/calmodulin-dependent protein kinase-mediated phosphorylation of neurofilament proteins and inhibition of calpain activity in the sciatic nerve in OPIDN. Proteinase inhibition has been reported recently to result in enhanced phosphorylation of neurofilaments in some cells. The present study suggests that the enhanced phosphorylation of neurofilaments by DFP-increased Ca2+/calmodulin-dependent protein kinase activity may be contributing toward alteration in NF axonal transport and the development of OPIDN.

Induction of cytochrome P450 isozymes by simultaneous inhalation exposure of hens to n-hexane and methyl iso-butyl ketone (MiBK).

Chickens were exposed simultaneously to the industrial hexacarbon solvents n-hexane and methyl iso-butyl ketone (MiBK). n-Hexane has been shown to be neurotoxic in both humans and other vertebrates. While MiBK is not neurotoxic, it has been shown to greatly synergize the clinical appearance of neurotoxicity in animals exposed to both of these solvents. Groups of hens were exposed for 29 days in inhalation chambers to 1000 ppm n-hexane in combination with 10, 100, 250, 500, or 1000 ppm MiBK. Other groups received either 1000 ppm n-hexane, 1000 ppm MiBK, or ambient air and served as controls. A dose-dependent decrease in body weight and an increase in clinical effects were noted for the highest exposure groups (1000 ppm n-hexane combined with 1000, 500 or 250 ppm MiBK). There was an MiBK dose-dependent increase in cytochrome P450 content and benzphetamine N-demethylase activity, but there was no distinct pattern for ethoxyresorufin O-deethylase or cytochrome c reductase activities. Mixed-function oxidase levels and activities (cytochrome P450 content and benzphetamine N-demethylase) were elevated significantly (P less than 0.05) over controls even in the lowest MiBK group (10 ppm), although there were no clinical signs of neurotoxicity. Four different isozymes of cytochrome P450 were measured immunologically. There was a dose-dependent increase in three of the isozymes, two of which were phenobarbital inducible and one of which was induced by beta-napthoflavone. Quantitatively, the largest increase was in the PB-A isozyme, a phenobarbital-inducible isozyme which accounted for approximately 70% of the cytochrome P450 present in animals treated with MiBK. The results suggest that MiBK selectively induces cytochrome P450 isozymes leading to the metabolic activation of the weak neurotoxicant n-hexane to the potent neurotoxicant 2,5-hexanedione (2,5-HD).

Comparison of endogenous phosphorylation of hen and rat spinal cord proteins and partial characterization of optimal phosphorylation conditions for hen spinal cord.

The optimal conditions for the endogenous phosphorylation of hen spinal cord cytosolic and membrane proteins with 5 ?M [?-(32)P]ATP, 10 mM MgCl(2), were determined by 10% SDS-polyacrylamide gel electrophoresis, autoradiography, and microdensitometry. Phosphate incorporation increased linearly with concentrations ranging from 35-75 ?g/100 ?l for cytosolic proteins and 21-125 ?g/200 ?l for membrane proteins. Optimal incubation times, temperatures, and pH values were 60 s, 30 degrees C, and 6.0, respectively, for spinal cord cytosolic proteins and 15 s, 45 degrees C, and 8.0, respectively, for spinal cord membranes. Prominent species differences in protein phosphorylation between these fractions in hens and similarly prepared fractions in rats, co-electrophoresed, include 80K and 30K protein phosphate acceptors unique to rat spinal cord cytosol, 60K and 16K protein phosphate acceptors characteristic of rat spinal cord membranes, a 50K protein phosphate acceptor present only in hen spinal cord membranes, and greater phosphorylation of a more abundant 20K protein in both hen spinal cord fractions. The functional significance of these differences is presently unclear. However, their characterization provides a basis from which to launch future investigations of the biochemistry, pharmacology, and toxicology of spinal cord protein phosphorylation and indicates that caution should be exercised in the choice of an animal model with characteristics appropriate to those of the system it is representing.

Biochemical changes in sciatic nerve of hens treated with tri-o-cresyl phosphate: increased phosphorylation of cytoskeletal proteins.

Calcium- and calmodulin-regulated protein phosphorylation has been suggested to play a role in the pathogenesis of organophosphorus compound-induced delayed neurotoxicity (OPIDN). This condition is characterized by ataxia that progresses to paralysis concurrent with a central-peripheral distal axonopathy after a delay period of 1-2 weeks following exposure to an organophosphorus compound causing delayed neurotoxicity, such as tri-o-cresyl phosphate (TOCP). Calcium/calmodulin (CaM) kinase II is involved in the increased phosphorylation of brain microtubule and spinal cord neurofilament triplet proteins following treatment of animals with organophosphorus compounds that are capable of producing OPIDN. In this study, chickens were given a single oral neurotoxic dose of 750 mg TOCP/kg body weight and killed after 1, 6, 14 or 21 days following treatment. Protein kinase-mediated phosphorylation of cytoskeletal proteins was studied in proximal and distal parts of sciatic nerves of control and treated hens. Peripheral nerve proteins were phosphorylated in vitro using [gamma-32P]ATP as a phosphoryl group donor. Phosphorylated proteins were separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein phosphorylation was detected by autoradiography and quantified by laser microdensitometry. The extent of Ca2+-calmodulin dependent phosphorylation of five cytoskeletal proteins was significantly increased in TOCP treated animals, particularly at 1 and 6 days after treatment, in both the proximal and distal portion of the nerve. The identity of these proteins was confirmed by 2-D PAGE as tubulin, the neurofilament triplet proteins and microtubule associated protein-2 (MAP-2). These results confirm earlier observation of the close temporal relationship between increased cytoskeletal protein phosphorylation and the development and OPIDN.

In vivo binding of [(14)C]acrylamide to proteins in the mouse nervous system.

The labeling of mouse brain and spinal cord proteins in vivo following the administration of 200 ?g/kg [(14)C]acrylamide (25 mCi/kg, i.p.) was measured by polyacrylamide gel electrophoresis and autoradiography. Several cytoskeletal proteins had the highest specific activity. These included the medium (130 kDa) and high (180 kDa) molecular weight neurofilament proteins and unknown proteins with molecular weights of 31, 33 and 57 kDa. About 0.05% of the neurofilament proteins were labeled with the dose employed. Although accurate assessments of the stoichiometry for acrylamide binding to 31, 33 and 57 kDa bands could not be made due to the low concentrations of these proteins, it is estimated that over 0.1% of these proteins bound acrylamide. There were significant amounts of acrylamide bound to proteins seven days after exposure, with labeling ranging from 15 to 60% of the levels observed one day after exposure. These results show that acrylamide binds to several major cytoskeletal proteins present in the mouse nervous system. The formation of these adducts may play a role in the neurotoxicity of acrylamide.

Acrylamide increases in vitro calcium and calmodulin-dependent kinase-mediated phosphorylation of rat brain and spinal cord neurofilament proteins.

Male Sprague-Dawley rats were administered a daily i.p. dose of 0.70 mmol/kg body weight of acrylamide, propionamide (a non-neurotoxic structural analog of acrylamide) or deionized water. Animals were sacrificed when signs of severe neurotoxicity were apparent. Neurofilaments (NFs) and endogenous kinase were isolated from the brain and spinal cord by axonal floatation. Increased in vitro Ca2+/calmodulin-dependent phosphorylation of endogenous and exogenous NF proteins and autophosphorylation of Ca2+/calmodulin protein kinase II (CaM kinase II, EC 2-7-1-37) were observed in samples from both brain and spinal cord of acrylamide-treated animals compared with controls. There was no significant difference between samples isolated from propionamide-treated animals and controls. Increased calmodulin binding to brain supernatant CaM kinase II was also observed as a result of acrylamide treatment. There was no significant difference observed in the amount of antibody binding to the alpha-subunit of brain supernatant CaM kinase II between treated or control animals. These results suggest that increased CaM kinase II-dependent phosphorylation of cytoskeletal proteins may be involved in the mechanisms of acrylamide-induced neurotoxicity.

Enhanced calmodulin binding concurrent with increased kinase-dependent phosphorylation of cytoskeletal proteins following a single subcutaneous injection of diisopropyl phosphorofluoridate in hens.

Diisopropyl phosphorofluoridate (DFP) produces Type I organophosphorus compound-induced delayed neurotoxicity (OPIDN) in adult female chickens. We have proposed that calcium/calmodulin protein kinase II (CaM kinase II) plays a role in the development of OPIDN by increasing the phosphorylation of cytoskeletal proteins. We investigated in vivo the effects of treatment of DFP on CaM kinase II-dependent phosphorylation. In isolated brain supernatants from DFP-treated hens, calmodulin binding increased concurrent with increases in CaM kinase II-dependent autophosphorylation and phosphorylation of cytoskeleton proteins. There were no changes in the relative amounts of the enzyme based on immunobinding studies of antibodies to the CaM kinase II. In the absence of any exogenously added substrate. CaM kinase II and microtubule associated protein-2 (MAP-2) exhibited substantially increased phosphorylation, 833 and 275%, respectively, over brain supernatants from untreated hens. Moreover, isolated brain supernatants from treated hens with exogenously added cytoskeletal proteins and myelin basic protein (MBP) exhibited significant increases in phosphorylation over control, 233, 332 and 60%, for MAP-2, tubulin, and MBP, respectively. 125I-Calmodulin binding studies revealed a 136% increase in calmodulin binding to CaM kinase II in treated hens when compared to control groups. The data suggest that in vivo DFP treatment increases the percentage of unphosphorylated, active CaM kinase II resulting in increased calmodulin binding and subsequent enhanced phosphorylation of cytoskeletal proteins that leads to their aggregation and the production of axonal degeneration.

Placental transfer and pharmacokinetics of a single dermal dose of [14C]methyl parathion in rats.

The pharmacokinetics and placental transfer of a single dermal 10.0 mg (10microCi)/kg dose of uniformly phenyl-labeled [14C] methyl parathion (0,0-dimethyl 0-4-nitrophenyl phosphorothioate) were investigated in pregnant Sprague-Dawley rats at 14-18 days of gestation. Three rats were killed at each time interval: 1, 2, 4, 12, 24, 48, 72, and 96 h after dosing. Radioactivity disappeared biexponentially from the administration sites, which retained 50% and 3% of the dose after 1 h and 96 h, respectively. Most of the absorbed radioactivity was excreted in the urine (91%). Only 3% of the 14C was recovered in the feces. One h after the administration, radioactivity was detected in all tissues, including fetal tissue. The peak maternal plasma concentration of radioactivity (ng methyl parathion equivalent/ml) was 1005 at 2 h, compared to 318 ng for fetal plasma at 12 h. The maximum concentrations of radioactivity (ng methyl parathion equivalent/g), detected in most tissues within 12 h of dosing, were, in descending order: adipose tissue (67,532), kidney (1,571), spleen (1,256), spinal cord (1,004), heart (729), liver (706), brain (546), placenta (389), and fetus (256). The metabolism studies showed that methyl parathion, detected by HPLC, was the major compound identified in plasma and tissues. The maximum concentration detected was in plasma, at 513 ng/ml, and in the following tissues (ng/g fresh tissue): kidney (819), fetus (668), placenta (394), liver (375), and brain (282). The metabolite methyl paraoxon was detected in maternal brain and liver at maximum concentrations (ng/g fresh tissue) of 135 and 64 after 12 h and 4 h respectively, while p-nitrophenol was only detected in liver at a maximum concentration of 21 ng/g 72 h after dosing. Pharmacokinetic studies showed that methyl parathion disappeared monoexponentially from plasma and tissues. The half-life of elimination of methyl parathion from plasma was 11 h corresponding to a constant rate value of 0.06 h(-1). The results indicate that skin and placenta are poor barriers against methyl parathion permeability, resulting in a rapid and extensive dermal absorption of this insecticide and extensive placental transfer. This is indicated by the relative residence (R(R)) of methyl parathion in the plasma, which was largest in the placenta followed by the fetus. This study suggests that pregnant women and fetuses may be at risk of cholinergic toxicity following dermal exposure to methyl parathion.

Acute sarin exposure causes differential regulation of choline acetyltransferase, acetylcholinesterase, and acetylcholine receptors in the central nervous system of the rat.

Acute neurotoxic effects of sarin (O:-isopropylmethylphosphonoflouridate) in male Sprague-Dawley rats were studied. The animals were treated with intramuscular (im) injections of either 1 x LD(50) (100 microg/kg), and sacrificed at 0. 5, 1, 3, 6, 15, or 20 h after treatment, or with im injections of either 0.01, 0.1, 0.5, or 1 x LD(50) and sacrificed 15 h after treatment. Plasma butyrylcholinesterase (BChE) and brain regional acetylcholinesterase (AChE) were inhibited (45-55%) by 30 min after the LD(50) dose. BChE in the plasma and AChE in cortex, brainstem, midbrain, and cerebellum remained inhibited for up to 20 h following a single LD(50) treatment. No inhibition in plasma BChE activity was observed 20 h after treatment with doses lower than the LD(50) dose. Midbrain and brainstem seem to be most responsive to sarin treatment at lower doses, as these regions exhibited inhibition (approximately 49% and 10%, respectively) in AChE activity following 0.1 x LD(50) treatment, after 20 h. Choline acetyltransferase (ChAT) activity was increased in cortex, brainstem, and midbrain 6 h after LD(50) treatment, and the elevated enzyme activity persisted up to 20 h after treatment. Cortex ChAT activity was significantly increased following a 0.1 x LD(50) dose, whereas brainstem and midbrain did not show any effect at lower doses. Treatment with an LD(50) dose caused a biphasic response in cortical nicotinic acetylcholine receptor (nAChR) and muscarinic acetylcholine receptor (m2-mAChR) ligand binding, using [(3)H]cytisine and [(3)H]AFDX-384 as ligands for nAChR and mAChR, respectively. Decreases at 1 and 3 h and consistent increases at 6, 15, and 20 h in nAChR and m2-mAChR were observed following a single LD(50) dose. The increase in nAChR ligand binding densities was much more pronounced than in mAChR. These results suggest that a single exposure of sarin, ranging from 0.1 to 1 x LD(50), modulates the cholinergic pathways differently and thereby causes dysregulation in excitatory neurotransmission.

Locomotor and sensorimotor performance deficit in rats following exposure to pyridostigmine bromide, DEET, and permethrin, alone and in combination.

Since their return from Persian Gulf War (PGW), many veterans have complained of symptoms including muscle and joint pain, ataxia, chronic fatigue, headache, and difficulty with concentration. The causes of the symptoms remain unknown. Because these veterans were exposed to a combination of chemicals including pyridostigmine bromide (PB), DEET, and permethrin, we investigated the effects of these agents, alone and in combination, on the sensorimotor behavior and central cholinergic system of rats. Male Sprague-Dawley rats (200-250 gm) were treated with DEET (40 mg/kg, dermal) or permethrin (0.13 mg/kg, dermal), alone and in combination with PB (1.3 mg/kg, oral, last 15 days only), for 45 days. Sensorimotor ability was assessed by a battery of behavioral tests that included beam-walk score, beam-walk time, incline plane performance, and forepaw grip on days 30 and 45 following the treatment. On day 45 the animals were sacrificed, and plasma and CNS cholinesterase, and brain choline acetyl transferase, muscarinic and nicotinic acetylcholine receptors were evaluated. Animals treated with PB, alone or in combination with DEET and permethrin, showed a significant deficit in beam-walk score as well as beam-walk time as compared with controls. Treatment with either DEET or permethrin, alone or in combination with each other, did not have a significant effect on beam-walk score. All chemicals, alone or in combination, resulted in a significant impairment in incline plane testing on days 30 and 45 following treatment. Treatment with PB, DEET, or permethrin alone did not have any inhibitory effect on plasma or brain cholinesterase activities, except that PB alone caused moderate inhibition in midbrain acetylcholinesterase (AChE) activity. Treatment with permethrin alone caused significant increase in cortical and cerebellar AChE activity. A combination of DEET and permethrin or PB and DEET led to significant decrease in AChE activity in brainstem and midbrain and brainstem, respectively. A significant decrease in brainstem AChE activity was observed following combined exposure to PB and permethrin. Coexposure with PB, DEET, and permethrin resulted in significant inhibition in AChE in brainstem and midbrain. No effect was observed on choline acetyl transferase activity in brainstem or cortex, except combined exposure to PB, DEET, and permethrin caused a slight but significant increase in cortical choline acetyltransferase activity. Treatment with PB, DEET, and permethrin alone caused a significant increase in ligand binding for m2 muscarinic acetylcholine receptor (mAChR) in the cortex. Coexposure to PB, DEET, and permethrin did not have any effect over that of PB-induced increase in ligand binding. There was no significant change in ligand binding for nicotinic acetylcholine receptor (nAChR) associated with treatment with the chemical alone; a combination of PB and DEET or coexposure with PB, DEET, and permethrin caused a significant increase in nAChR ligand binding in the cortex. Thus, these results suggest that exposure to physiologically relevant doses of PB, DEET, and permethrin, alone or in combination, leads to neurobehavioral deficits and region-specific alterations in AChE and acetylcholine receptors.

Diisopropyl phosphorofluoridate (DFP) treatment alters calcium-activated proteinase activity and cytoskeletal proteins of the hen sciatic nerve.

Diisopropyl phosphorofluoridate (DFP) produces delayed neurotoxicity (OPIDN) in hens that is characterized by peripheral and central axonal degeneration. DFP administration resulted in mCANP activity inhibition in sciatic nerve and significant decrease in total NF-H, phosphorylated NF-H, vimentin, GFAP, tubulin, and tau. The degradation of cytoskeletal proteins even in the presence of decreased CANP activity may be ascribed to the release of intracellular Ca2+, elevation of other proteinase activity, or modification of cytoskeletal proteins resulting in their increased susceptibility in OPIDN.