DFP (Diisopropyl fluorophosphate)
CAS No. 55-91-4
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ACTIVITY: Former Insecticide (Organophosphate)


•• See 79 Reports available from The National Technical Information Service

DFP is a structural analog of sarin.
Ref: Sarin (nerve agent GB)-induced differential expression of mRNA coding for the acetylcholinesterase gene in the rat central nervous system; by Damodaran TV, Jones KH, Patel AG, Abou-Donia MB. Biochemical Pharmacology Volume 65, Issue 12 , 15 June 2003, Pages 2041-2047.

From Science Direct

Environmental Toxicology and Pharmacology Volume 7, Issue 2 , April 1999, Pages 147-152

Organophosphate-induced brain injuries: delayed apoptosis mediated by nitric oxide

Yun-Bae Kim (a), Gyeung-Haeng Hur (a), Sungho Shin (a), Dai-Eun Sok (b), Jong-Koo Kang (c) and Yong-Soon Lee (d)

(a) Biomedical Assessment Laboratory (1-3-4), Agency for Defense Development, Yuseong P.O. Box 35-1, Taejon 305-600, South Korea
(b) College of Pharmacy, Chungnam National University, Taejon 305-764, South Korea
(c) College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, South Korea
(d) College of Veterinary Medicine, Seoul National University, Suwon 441-744, South Korea

The features of organophosphate-induced brain injuries were investigated. Rats were poisoned intraperitoneally with 9 mg/kg (1.8 LD50) of diisopropylfluorophosphate [synonym for DFP] . Pyridostigmine bromide (0.1 mg/kg) and atropine methylnitrate (20 mg/kg), which are centrally inactive, were pre-treated intramuscularly to reduce the mortality and eliminate peripheral signs. Diisopropylfluorophosphate induced severe limbic seizures, and early necrotic and delayed apoptotic brain injuries. The necrotic brain injury was observed to be maximal as early as 1 h after diisopropylfluorophosphate treatment predominently in hippocampus and piriform/entorhinal cortices, showing a spongiform change (malacia) of neuropils in severe cases. In contrast, typical apoptotic (TUNEL-positive) cells started to appear at 12 h in thalamus, and a mixed type in amygdala. Separately, nitrite/nitrate content in cerebrospinal fluid was found to significantly increase after 2 h, reaching a maximal level at 6 h. Pre-treatment with -NG-nitroarginine, an inhibitor of nitric oxide synthase, reduced nitrite/nitrate content and, noteworthy, attenuated only apoptotic brain injury in all four brain regions without affecting seizure intensity and necrotic injury. Taken together, the delayed apoptotic injury of brain induced by diisopropylfluorophosphate poisoning in rats might be mediated in part through nitric oxide production.

Neurochemical Research 25 (6): 809-816, June 2000

Alterations in Levels of mRNAs Coding for Glial Fibrillary Acidic Protein (GFAP) and Vimentin Genes in the Central Nervous System of Hens Treated with Diisopropyl Phosphorofluoridate (DFP)

Tirupapuliyur V. Damodaran, Mohamed B. Abou-Donia

Department of Pharmacology and Cancer Biology, Duke University Medical Center,Durham,North Carolina

Diisopropyl phophorofluoridate (DFP) produces organophosphorus-ester induced delayed neurotoxicity (OPIDN) in the hen, human and other sensitive species. We studied the effect of DFP admimistration (1.7 mg/kg/s.c.) on the expression of Intermediate Filament (IF) proteins: Glial Fibrillary Acidic Protein (GFAP) and vimentin which are known indicators of neurotoxicity and astroglial pathology. The hens were sacrificed at different time points i.e. 1,2,5,10 and 20 days. Total RNA was extracted from the following brain regions: cerebrum, cerebellum, and brainstem as well as spinal cord. Northern blots prepared using standard protocols were hybridized with GFAP and vimentin as well as [beta]-actin and 18S RNA cDNA (controls) probes. The results indicate a differential/spatial/temporal regulation of GFAP and vimentin levels which may be due to the result of disruption of glial-neuronal network. The GFAP transcript levels reached near control levels (88% and 95%) at 20 days post DFP treatment after an initial down-regulation (60% and 73%) in highly susceptible tissues like spinal cord and brainstem respectively. However vimentin transcript levels remained down-regulated (61% and 53%) at 20 days after an early reduced levels(47% and 55%) for spinal cord and brainstem respectively. This may be due to the astroglial pathology resulting in neuronal alterations or vice-versa. In cerebellum (less susceptile tissue) GFAP levels were moderately down-regulated at 1,2 and 5 days and reached near control values at 10 and 20 days. Vimentin was rapidly reinduced (128%) in cerebellum at 5 days and remained at the same level at 10 days and then returned to control values at 20 days after an initial down-regulation at 1 and 2 days. Thus these alterations were less drastic in cerebellum as indicated by initial susceptibility followed by rapid recovery. On the other hand both GFAP and vimentin levels were upregulated from 2 days onwards in the non-susceptible tissue cerebrum, implying protective mechanisms from the beginning. Hence the DFP induced astroglial pathology as indicated by the complex expression profile of GFAP and vimentin mRNA levels may be playing an important role in the delayed degeneration of axons or is the result of progressive degeneration of axons in OPIDN.

Note from FAN. This may help you understand "astroglial"
"The astroglial cell mass constitutes a prominent part of the total brain cell number and volume (93). The anatomy of these cells, which "float" in the extracellular space of the brain, caused Virchow to propose, more than 100 years ago, that they have a metabolic and structurally supportive role for the neurons. Since then and until the last 15–20 years, this supportive role has been considered as passive and solely dependent on the requirements of the degree of neuronal activity. Very little attention has been paid to the astroglial cells for decades, mostly owing to the difficulties of studying them in the intact nervous system. Another important reason for this problem has been the lack of markers for the identification of the cells. Neurons were relatively easy to identify using electrophysiological techniques, mostly owing to their action potentials. Astroglial cells, on the other hand, are small, with cell bodies measuring 10-15 mm in diameter. The cell membrane can be depolarised but no action potential can be elicited. Furthermore, due to the syncytial arrangement, with the cells being electrically coupled to each other, it has been difficult to use conventional electrophysiology to register ion currents from the cells (see below). Therefore, it was not until the early 1970s that Eng, Bignami and co-workers (30) identified and isolated the glial fibrillary acidic protein (GFAP) as a true marker for the cells.
At the same time, methods were developed for the cultivation of astroglial cells. Earlier, cell lineages of tumor-derived glial cells had been cultivated, but a problem with those cells was the relatively low degree of differentiation, which made it difficult to extrapolate results from these experiments to the in vivo situation. Using different primary cultures, astroglial cells could be enriched and characterized in large numbers (11,42,79). Since then, astroglial research has developed very rapidly. In addition to their utility functions in relation to the neurons (e.g., energy supply, removal of extracellular K+ and Glu, and synthesis and release of neurotrophic factors) the cells have the capacity to monitor synaptic activity, to sense the composition of the extracellular space and the blood serum, to integrate the information obtained, and to influence neuronal activity by regulating the extracellular ion and amino acid homeostasis and the concentration and composition of trophic substances (Fig. 1). ..."
Ref: Astroglial Pharmacology by Elisabeth Hansson et al. Paper available online at http://www.acnp.org/g4/GN401000079/Ch079.html

Neurochemical Research 25 (12): 1579-1586, December 2000

Early Differential Induction of C-jun in the Central Nervous System of Hens Treated with Diisopropylphosphorofluoridate (DFP)

Tirupapuliyur V. Damodaran, Ali Abdel Rahman, Mohamed B. Abou-Donia

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus-ester induced delayed neurotoxicity (OPIDN) in the hen, human and other sensitive species. We studied the effect of a single dose of DFP (1.7 mg/kg/sc) on the expression of c-jun, which is one of the heterodimerizing ITFs (Inducible Transcriptional Factors) of the AP-1 family. The hens were sacrificed at different time points ie 0.25,.0.50, 1 and 2 hrs. Total RNA was extracted from the following brain regions: cerebrum, cerebellum, brainstem, midbrain and as well as spinal cord. Northern blots prepared using standard protocols were hybridized with c-jun as well as b-actin and 18S RNA cDNA (control) probes. The results indicate differential regulation of c-jun levels which may be due to the activation of both cholinergic and non-cholinergic pathways of CNS, besides changing roles of c-jun (as mediator of degeneration or regeneration) depending on heterodimerization with other ITFs. In the highly susceptible tissues like brainstem and spinal cord c-jun transcript levels increased at 15 minutes and continued to increase gradually till it reached the maximum at 2 hrs. Overall spinal cord showed the maximum levels of c-jun induction (207%) at 2 hrs time point of all the CNS tissues. The enhancement of cholinergic transmisson by the inhibition of cholinestrase may be responsible for the gradual increase mediated by neural and vascular factors. In contrast, less susceptible tissue, cerebellum showed almost immediate induction to high level of (179%) at 15 minutes and the levels stayed more or less the same until it peaked to 185% at 2 hrs. Relatively low abundance of cholinergic neurons and high number of sensitized specialized cell types like Bergman glia and Purkinje cells may be responsible for the immediate higher induction. Non-susceptible tissue cerebrum did not show any changes in the c-jun levels. In midbrain the induction pattern was very similar to that of brainstem. This differential induction pattern of c-jun encomposing the differences in the quantity and time course was directly proportionate to the degree of susceptibility and cellular heterogeneity of different regions of CNS. The significant increase in c-jun levels along with our earlier observation on the increased c-fos levels indicate that AP-1 family of genes may be one of the IEGs involved in the long term changes which eventually lead to OPIDN.

From Dart Special at Toxnet

DEV NEUROSCI 5:508-519,1982



Taxonomic Name: RATTUS, WISTAR

Test Object: MAMMAL, RAT
Sex Treated: FEMALE

Name of Agent (CAS RN):
ARACHIS OIL ( 8002-03-7 )

Assay Method:



Chem. Res. Toxicol., 19 (2), 334 -339, 2006.

Aging of Mipafox-Inhibited Human Acetylcholinesterase Proceeds by Displacement of Both Isopropylamine Groups to Yield a Phosphate Adduct

Timothy J. Kropp and Rudy J. Richardson*

Toxicology Program, Department of Environmental Health Sciences, University of Michigan, 1420 Washington Heights, Ann Arbor, Michigan 48109-2029

Aging of phosphylated serine esterases, e.g., acetylcholinesterase (AChE) and neuropathy target esterase (NTE), renders the inhibited enzymes refractory to reactivation. This process has been considered to require postinhibitory side group loss from the organophosphorus moiety. Recently, however, it has been shown that the catalytic domain of human NTE inhibited by N,N'-diisopropylphosphorodiamidofluoridate (mipafox, MIP) ages by deprotonation. For mechanistic understanding and biomarker development, it would be important to know the identity of the MIP adduct on target esterases after inhibition and aging occurred. Accordingly, the present study was performed to determine if MIP-inhibited human AChE ages by side group loss or an alternate method, e.g., deprotonation. Diisopropylphosphorofluoridate (DFP), the oxygen analogue of MIP, was used for comparison, because DFP-inhibited AChE is known to age by net loss of an isopropyl group. Kinetics experiments were done with DFP and MIP against AChE to follow the time course of inhibition, reactivation, and aging for each inhibitor. MS studies of tryptic digests from kinetically aged DFP-inhibited AChE revealed a mass shift of 122.8 ± 0.7 Da for the active site peptide (ASP) peak, corresponding to the expected monoisopropylphosphoryl adduct. In contrast, the analogous mass shift for kinetically aged MIP-inhibited AChE was 80.7 ± 0.9 Da, corresponding to a phosphate adduct. Because this finding was unexpected, the identity of the phosphoserine-containing ASP was confirmed by immunoprecipitation followed by MS. The results indicate that aging of MIP-inhibited AChE proceeds by displacement of both isopropylamine groups. Further research will be required to elucidate the detailed mechanism of formation of a phosphate conjugate from MIP-inhibited AChE; however, knowledge of the identity of this adduct will be useful in biomarker studies.


Brain Res 2003 Mar 7;965(1-2):180-6

The cholinesterase inhibitor DFP facilitates the expression of paradoxical sleep (PS) propensity in rats subjected to short-term PS deprivation.

Deurveilher S, Hennevin E.

Laboratoire de Neurobiologie de l'Apprentissage, de la Memoire et de la Communication, UMR CNRS 8620, Universite Paris-Sud, Bat. 446, 91405 Cedex, Orsay, France

Short-term paradoxical sleep (PS) deprivation was used to examine the effects of chronic exposure to subtoxic doses of the cholinesterase inhibitor diisopropylfluorophosphate (DFP) on PS regulation. Rats were injected once daily with DFP (0.2 mg/kg per day; s.c.) for 11 consecutive days; control rats received a daily injection of oil vehicle. The experiment was conducted on the 10th and 11th days of treatment, when brain cholinesterase inhibition induced by DFP exposure was maximal. On the 10th day, an 8-h baseline recording was carried out. On the 11th day, a 6-h PS deprivation was carried out by manually awaking rats each time they showed polygraphic signs of PS; recordings were then continued for another 2 h to examine recovery sleep. During deprivation, though they slept less than controls, DFP-treated rats made more attempts to enter PS. After deprivation, their PS rebound had an overall amount comparable to that of the controls, but its time course was shortened: whereas PS elevation was manifested through the 2 h of recovery in the control group, it occurred only during the first hour in the DFP group. These results demonstrate that chronic, low-level DFP exposure facilitated the expression of the PS propensity that accumulated as a result of PS deprivation: it enhanced the tendency for PS during deprivation; it accelerated the rate of compensatory PS expression after deprivation. They support the hypothesis that DFP promotes PS initiation by increasing cholinergic transmission.

PMID: 12591136 [PubMed - in process]


2002 Neurochem Res Mar;27(3):183-93

Early differential elevation and persistence of phosphorylated cAMP-response element binding protein (p-CREB) in the central nervous system of hens treated with diisopropyl phosphorofluoridate, an OPIDN-causing compound.

Damodaran TV, Abdel-Rahman AA, Suliman HB, Abou-Donia MB.

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27708, USA.

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus-ester-induced delayed neurotoxicity in sensitive species. We studied the effect of single dose of DFP on the expression of phosphorylated cAMP-response element binding protein (p-CREB), which is a well known transcription factor involved in several pathways mediating different types of external stimuli. The hens were perfused with neutral buffered formalin at different time points, i.e., 0.5, 1.0, and 2.0 hrs, as well as 1, 2, 5, and 20 days after dosing. The central nervous system regions of the whole brain were dissected and 7-micron sections were stained for either p-CREB immunopositivity or with hematoxylin and eosin. Results indicated an early differential increase of p-CREB immunopositivity in susceptible regions such as cerebellum, brainstem, and midbrain within 2 hrs. These induced levels persisted upto 5 days in these tissues, although the time course of p-CREB immunopositivity was distinctly different for each region. In the cerebellum induction of p-CREB was seen in the granular layer where both the granulocytes and the glial cells showed induction. Increased immunopositivity for p-CREB in the Purkinje cells and in some basket cells of the molecular layer was noticed over time, but the induction was not as great as in the granular layer. Of all the tissues cerebellum showed the strongest intensity of immunopositivity of the cells as well as the highest (absolute) number of pCREB-positive cells. The brainstem showed a similar fluctuating pattern like the cerebellum with the highest percentage increase of the immunoreactive cells at 5 days preceded by the lowest dip in immunopositivity at 2 days. In the midbrain, there was a time-dependent increase in the immunopositivity from 0.5 hr onwards until reaching control levels at 20 days. Immunopositivity was also noted in portions of the spina medularis and spina oblongata. The cerebrum (non-susceptible tissue) of DFP-treated hens did not show much deviation from the controls. The endothelial cells of the susceptible regions showed induction at early time points, in contrast to the absence of induction in cerebrum. Spatial and temporal differences in the immunopositivity pattern indicate probable involvement of CREB-independent pathways also. Overall, the complex induction pattern of p-CREB, along with our earlier observations of the early induction of c-fos, c-jun and Protein Kinase A (PKA) as well as the induction of Calcium2+/Calmodulin dependent Protein Kinase II (CaM kinase II) at later periods, strongly suggest an activator role of CREB mediated pathways that may lead to the clinical development of delayed neurotoxicity.

PMID: 11958515 [PubMed - indexed for MEDLINE]


2002 Biochem Pharmacol Jan 1;63(1):11-9

Protein levels of neurofilament subunits in the hen central nervous system following prevention and potentiation of diisopropyl phosphorofluoridate (DFP)-induced delayed neurotoxicity(1).

Xie K, Gupta RP, Abou-Donia MB.

Neurotoxicology Laboratory, School of Life Science, University of Science and Technology of China, 230027, Hefei, Anhui, P. R. China.

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester, which produces delayed neurotoxicity (OPIDN) in hens in 7-14 days. OPIDN is characterized by mild ataxia in its initial stages and severe ataxia or paralysis in about 3 weeks. It is marked by distal swollen axons, and exhibits aggregations of neurofilaments (NFs), microtubules, proliferated smooth endoplasmic reticulum, and multivesicular bodies. These aggregations subsequently undergo disintegration, leaving empty varicosities. Previous studies in this laboratory have shown an increased level of medium-molecular weight NF (NF-M) and decreased levels of high- and low-molecular weight NF (NF-H, NF-L) proteins in the spinal cord of DFP-treated hens. The main objective of this investigation was to study the effect of DFP administration on NF subunit levels when OPIDN is prevented or potentiated by pretreatment or post-treatment with phenylmethylsulfonyl fluoride (PMSF), respectively. Hens pretreated or post-treated with PMSF were killed 1, 5, 10, and 20 days after the last treatment. The alteration in NF subunit protein levels observed in DFP-treated hen spinal cords was not observed in protected hens. Estimation of NFs in the potentiation experiments, however, showed a different pattern of alteration in NF subunit levels. The results showed that an alteration in NF subunit levels in DFP-treated hens might be related to the development of OPIDN, since these changes were suppressed in PMSF-protected hens. However, results from PMSF post-treated hen spinal cords suggested that potentiation of OPIDN by PMSF was mediated by a mechanism different from that followed by DFP alone to produce OPIDN.

PMID: 11754869 [PubMed - indexed for MEDLINE]


2001 Arch Toxicol Aug;75(6):346-56

Nitric oxide modulates high-energy phosphates in brain regions of rats intoxicated with diisopropylphosphorofluoridate or carbofuran: prevention by N-tert-butyl-alpha-phenylnitrone or vitamin E.

Gupta RC, Milatovic D, Dettbarn WD.

Toxicology Department, Murray State University, Breathitt Veterinary Center, Hopkinsville, KY 42241-2000, USA. ramesh.gupta@murraystate.edu

Acute effects of seizure-inducing doses of the organophosphate compound diisopropylphosphorofluoridate (DFP, 1.25 mg/kg s.c.) or the carbamate insecticide carbofuran (CF, 1.25 mg/kg s.c.) on nitric oxide (NO) were studied in the brain of rats. Brain regions (pyriform cortex, amygdala, and hippocampus) were assayed for citrulline as the determinant of NO and for high-energy phosphates (ATP and phosphocreatine) as well as their major metabolites (ADP, AMP, and creatine). Rats, anesthetized with sodium pentobarbital (50 mg/kg i.p.), were killed using a head-focused microwave (power, 10 kW; duration, 1.7 s). Analyses of brain regions of controls revealed significantly higher levels of citrulline in the amygdala (289.8+/-7.0 nmol/g), followed by the hippocampus (253.8+/-5.5 nmol/g), and cortex (121.7+/-4.3 nmol/g). Levels of energy metabolites were significantly higher in cortex than in amygdala or hippocampus. Within 5 min of CF injection, the citrulline levels were markedly elevated in all three brain regions examined, while with DFP treatment, only the cortex levels were elevated at this time. With either acetylcholinesterase (AChE) inhibitor, the maximum increase in citrulline levels was noted 30 min post-injection (> 6- to 7-fold in the cortex, and > 3- to 4-fold in the amygdala or hippocampus). Within 1 h following DFP or CF injection, marked declines in ATP (36-60%) and phosphocreatine (28-53%) were seen. Total adenine nucleotides and total creatine compounds were reduced (36 58% and 28-48%, respectively). The inverse relationship between the increase in NO and the decease in high-energy phosphates, could partly be due to NO-induced impaired mitochondrial respiration leading to depletion of energy metabolites. Pretreatment of rats with an antioxidant, the spin trapping agent N-tert-butyl-alpha-phenylnitrone (PBN, 200 mg/kg i.p.), prevented DFP- or CF-induced seizures, while the antioxidant vitamin E (100 mg/kg i.p. per day for 3 days) had no anticonvulsant effect. Both antioxidants, however, significantly prevented the increase of citrulline and the depletion of high-energy phosphates. It is concluded that seizures induced by DFP and CF produce oxidative stress due to a marked increase in NO, causing mitochondrial dysfunction, and thereby depleting neuronal energy metabolites. PBN pretreatment provides protection against AChE inhibitor-induced oxidative stress mainly by preventing seizures. Additional antioxidant actions of PBN may contribute to its protective effects. Vitamin E has direct antioxidant effects by preventing excessive NO production.

PMID: 11570692 [PubMed - indexed for MEDLINE]


Biochem Cell Biol 2001;79(2):207-17

Effect of prevention and potentiation of diisopropyl phosphorofluoridate (DFP)-induced delayed neurotoxicity on the mRNA expression of neurofilament subunits in hen central nervous system.

Xie K, Gupta RP, Abou-Donia MB.

Neurotoxicology Laboratory, School of Life Science, University of Science and Technology of China, Hefei, Anhui, P.R. China.

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester, which produces mild ataxia in 7-14 days and severe ataxia or paralysis in about 20 days (OPIDN) in hens. Previous studies in this laboratory have shown enhanced temporal expression of neurofilament (NF) subunit mRNAs in the spinal cord (SC) of DFP-treated hens. The main objective of this investigation was to study the effect of DFP administration on NF subunit mRNAs expression, when OPIDN is protected or potentiated by pre-treatment or post-treatment, respectively, with phenylmethylsulfonyl fluoride (PMSF). The hens were sacrificed 1, 5, 10, and 20 days after the last treatment. In contrast with enhanced mRNA expression of NF subunits reported in OPIDN, there was no alteration in the expression of NF subunits in the SC of PMSF-protected hens that did not develop OPIDN. PMSF post-treatment of DFP-treated hens, which enhanced delayed neurotoxicity produced by a low dose of DFP, exhibited decrease in the mRNA expression of NF subunits in SC at all time periods (1-20 days) of observation. The expression of NF subunits was also studied in the degeneration-resistant tissue cerebrum of treated hens. The results from protected hens suggested that temporal enhanced expression of NF subunit mRNAs in DFP-treated hens might be contributing to the development of OPIDN in hens. By contrast, PMSF post-treatment seemed to potentiate OPIDN by a mechanism different from that followed by DFP alone to produce OPIDN.

PMID: 11310568 [PubMed - indexed for MEDLINE]


Neurochem Res 2001 Mar;26(3):235-43

Alteration in cytoskeletal protein levels in sciatic nerve on post-treatment of diisopropyl phosphorofluoridate (DFP)-treated hen with phenylmethylsulfonyl fluoride.

Xie K, Gupta RP, Abou-Donia MB.

Neurotoxicology Laboratory, School of Life Science, University of Science and Technology of China, Hefei, Anhui, PR China.

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester, and a single dose (1.7 mg/kg, sc.) of this compound produces mild ataxia in hens in 7-14 days and a severe ataxia or paralysis (OPIDN) in three weeks. OPIDN is associated with axonal swelling and their degeneration. We have previously observed alteration in neurofilament (NF) protein levels in the spinal cord of DFP-treated hens. The main objective of this investigation was to study NF protein levels in the sciatic nerves (SN) of hens, in which OPIDN has been potentiated by phenylmethylsulfonyl fluoride (PMSF) post-treatment. PMSF is known to protect DFP-treated (1.7 mg/kg) hens from developing OPIDN if injected before, and potentiate OPIDN if injected after the administration of DFP (0.5 mg/kg). The potentiation of OPIDN was accompanied by earlier elevation of NF proteins in the SN particulate fraction. In contrast, SN supernatant fraction showed a transient fall in NF protein levels in potentiation OPIDN. Out of the two other cytoskeletal proteins (i.e., tubulin, tau) studied in this investigation, tubulin also showed earlier elevation in its level in the particulate fraction in potentiated OPIDN. The earlier elevation of NF protein levels in SN particulate fraction in potentiated OPIDN suggested the possible involvement of NFs in delayed neurotoxicity.

PMID: 11495547 [PubMed - indexed for MEDLINE]

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
Volume 1546, Issue 2 , 7 April 2001, Pages 312-324

Insights into the reaction mechanism of the diisopropyl fluorophosphatase from Loligo vulgaris by means of kinetic studies, chemical modification and site-directed mutagenesis

Judith Hartleib and Heinz Rüterjans,

Institute of Biophysical Chemistry, Johann Wolfgang Goethe University of Frankfurt/M., Marie-Curie-Strasse 9, 60439 Frankfurt/M., Germany

Kinetic measurements, chemical modification and site-directed mutagenesis have been employed to gain deeper insights into the reaction mechanism of the diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris. Analysis of the kinetics of diisopropyl fluorophosphate hydrolysis reveals optimal enzyme activity at pH ≥8, 35°C and an ionic strength of 500 mM NaCl, where kcat reaches a limiting value of 526 s-1. The pH rate profile shows that full catalytic activity requires the deprotonation of an ionizable group with an apparent pKa of 6.82, ?Hion of 42 kJ/mol and ?Sion of 9.8 J/mol K at 25°C. Chemical modification of aspartate, glutamate, cysteine, arginine, lysine and tyrosine residues indicates that these amino acids are not critical for catalysis. None of the six histidine residues present in DFPase reacts with diethyl pyrocarbonate (DEPC), suggesting that DEPC has no accessibility to the histidines. Therefore, all six histidine residues have been individually replaced by asparagine in order to identify residues participating in catalysis. Only substitution of H287 renders the enzyme catalytically almost inactive with a residual activity of approx. 4% compared to wild-type DFPase. The other histidine residues do not significantly influence the enzymatic activity, but H181 and H274 seem to have a stabilizing function. These results are indicative of a catalytic mechanism in which H287 acts as a general base catalyst activating a nucleophilic water molecule by the abstraction of a proton.

NeuroToxicology Volume 22, Issue 2 , April 2001, Pages 191-202

A Comparative Study of Binding Sites for Diisopropyl Phosphorofluoridate in Membrane and Cytosol Preparations from Spinal Cord and Brain of Hens

Ryo Kamata (1, 3), Shin-ya Saito (2), Tadahiko Suzuki (1), Tadashi Takewaki (3), Hisayoshi Kofujita (4), Michikazu Ota (4) and Haruo Kobayashi (1)

1 Department of Veterinary Pharmacology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Japan
2 Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan
3 United Graduate School of Veterinary Sciences, Gifu University 1-1 Yanagido, Gifu 501-1193, Japan
4 Department of Wood Science and Technology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Japan
Received 23 February 2000; accepted 19 October 2000 Available online 9 May 2001.

Biochemical events in the initiation of organophosphorus induced delayed neurotoxicity (OPIDN) are not well understood. To find new putative target(s) for OPIDN, we investigated the biochemical and pharmacological characteristics of [3H]diisopropyl phosphorofluoridate (DFP) binding to membrane and cytosol preparations from the brain and spinal cord of hens in vitro. [3H]DFP binding to both preparations was determined by the specific binding obtained by subtracting non-specific binding from total binding. The specific binding sites of [3H]DFP were found not only on membrane but also in cytosol. Kd values were higher and Bmax values were lower in cytosol than in membrane. Moreover, the Kd values in both membrane and cytosol preparations from spinal cord were lower than those of brain. The Bmax values in membrane and cytosol were similar between brain and spinal cord. The specific binding to both preparations was markedly displaced by unlabeled DFP. The specific binding of DFP to the membrane was highly or partly displaced by organophosphorus compounds (OPs) or a carbamate, respectively. However, both the OPs and the carbamate had considerably weaker blocking effects on the specific binding of DFP to cytosol. None of the compounds known to interact with neuropathy target esterase (NTE) had a strong blocking effect on the specific binding of DFP to either membrane or cytosol. These results show that the specific binding of DFP to the membrane may be binding with cholinesterase (ChE). However, cytosol, especially in spinal cord, may have DFP binding sites other than ChE and NTE.

NeuroToxicology Volume 22, Issue 2 , April 2001, Pages 203-214

Correlation of Binding Sites for Diisopropyl Phosphorofluoridate with Cholinesterase and Neuropathy Target Esterase in Membrane and Cytosol Preparations from Hen

Ryo Kamata (1, 3), Shin-ya Saito (2), Tadahiko Suzuki (1), Tadashi Takewaki (3) and Haruo Kobayashi (1)

1 Department of Veterinary Pharmacology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Japan
2 Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan
3 United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
Received 23 February 2000; accepted 19 October 2000 Available online 9 May 2001.

To find new putative target(s) for organophosphorus induced delayed neurotoxicity (OPIDN), we investigated the biochemical and pharmacological characteristics of [3H]diisopropyl phosphorofluoridate (DFP) binding to membrane and cytosol preparations from the brain and spinal cord of hens. Specific [3H]DFP binding was determined by subtracting non-specific binding from total binding. The binding sites of [3H]DFP, an organophosphate that induces OPIDN, were found not only on membrane but also in cytosol. Reduction of subsequent ex vivo specific [3H]DFP binding by in vivo pretreatment with unlabeled DFP was found in cytosol, not membrane. The reduced binding lasted to the onset of OPIDN, especially in spinal cord. These results suggest that the specific DFP binding sites in cytosol, rather than on membrane, are the most important with regard to the initiation of OPIDN. Inhibitors of cholinesterase (ChE) and neuropathy target esterase (NTE) other than DFP did not affect specific [3H]DFP binding to either membranes or cytosol in vivo. Additionally, inhibition of the activities of these esterases by these compounds was not consistent with either the degree of inhibition of the [3H]DFP binding or a time-dependent manner of OPIDN. These results suggest that DFP binding site(s) involved in the initiation of OPIDN may be different from the active sites of ChE and NTE.


Toxicol Appl Pharmacol 1999 Jun 15;157(3):222-6

Neurotoxic potentiation is related to a metabolic interaction between p-bromophenylacetylurea and phenylmethanesulfonyl fluoride.

Xu J, Lister T, Purcell WM, Ray DE.

Faculty of Applied Sciences, University of the West of England, Bristol, United Kingdom.

This study investigated the neurotoxic potentiation and metabolic interaction between p-bromophenylacetylurea (BPAU) and phenylmethanesulfonyl fluoride (PMSF). The results showed that F344 rats given two successive daily doses of 150 mg/kg BPAU developed a moderate degree of ataxia. When rats were coadministrated a single intraperitoneal dose of 100 mg/kg PMSF either 1 day before, or 4 h or 1 day after the two daily doses of BPAU, the severity of ataxia was significantly increased. No such effect was observed when PMSF was given 4 days after BPAU, although this time point was still prior to the development of the neuropathy. The enhancement or potentiation of neuropathy by PMSF was thus seen only at times when parent BPAU was present in the target tissues. A pharmacokinetic study showed that PMSF increased the concentrations of BPAU and its metabolite, N'-hydroxy-p-bromophenylacetylurea (M1), in tissues and decreased the concentration of the metabolite 4-(4-bromophenyl)-3-oxapyrrolidine-2,5-dione (M2) in serum. This indicated that PMSF inhibited the M2 pathway and more BPAU was metabolized via the M1 pathway. This increased both BPAU and M1 levels in tissues and hence would have increased BPAU-induced neurotoxicity. We conclude that PMSF does not need to act directly on target sites to potentiate BPAU-induced neurotoxicity, since its interference with BPAU metabolism was sufficient to account for the increase in BPAU neurotoxicity. Thus a metabolic interaction underlies the neurotoxic potentiation between these two compounds rather than the target site interaction seen between PMSF and neuropathic organophosphates. This study is the first to demonstrate that interference with the metabolism of BPAU is an important aspect of the potentiation of BPAU-induced neurotoxicity. Copyright 1999 Academic Press.

PMID: 10373406 [PubMed - indexed for MEDLINE]


J Toxicol Environ Health 1997 Aug 29;51(6):571-90

Potentiation of organophosphorus compound-induced delayed neurotoxicity (OPIDN) in the central and peripheral nervous system of the adult hen: distribution of axonal lesions.

Randall JC, Yano BL, Richardson RJ.

Department of Environmental and Industrial Health, School of Public Health, University of Michigan, Ann Arbor 48109-2029, USA.

Clinical manifestations of mild organophosphorus compound-induced delayed neurotoxicity (OPIDN) produced by diisopropylphosphorofluoridate (DFP) in adult hens are potentiated by posttreatment with phenylmethanesulfonyl fluoride (PMSF). The purpose of this study was to assess whether potentiation of mild OPIDN produces a pattern of axonal lesions in the central and peripheral nervous system similar to that seen in severe OPIDN. Groups of 6 hens each were given the following priming/challenge doses sc at 0 and 4 h, respectively: 0.20 ml/kg corn oil/0.50 ml/kg glycerol formal (GF) (control); 0.50 mg/kg DFP/GF (low-dose DFP); 0.50 mg/kg DFP/60 mg/kg PMSF (potentiated DFP); 60 mg/kg PMSF/GF (PMSF alone); 60 mg/kg PMSF/1.5 mg/kg DFP (protected DFP); and 1.5 mg/kg DFP/GF (high-dose DFP). Two hens from each group were used to assay brain neurotoxic esterase (NTE) 24 h after the challenge dose, and the remaining hens were scored for deficits in walking, standing, and perching ability on d 18. Three hens from each group were perfusion-fixed on d 22 and neural tissues were prepared for histologic evaluation. DFP and/or PMSF caused > 88% brain NTE inhibition in all treated groups, compared to control. Protected DFP yielded no clinical deficits and a distribution and frequency of axonal lesions similar to control. PMSF alone produced a small increase in the frequency of lesions in the cervical spinal cord and peripheral nerves compared to control. Low-dose DFP caused minimal ataxia and increased frequency of axonal lesions in dorsal and lateral cervical spinal cord, ventral lumbar spinal cord, and inferior cerebellar peduncles (ICP) compared to control. Potentiated DFP and high-dose DFP produced maximal ataxia and essentially identical increases in the frequency of lesions in dorsal and ventral thoracic spinal cord, lateral lumbar spinal cord, and peripheral nerves compared to low-dose DFP. The results indicate that PMSF potentiation of mild OPIDN induced in adult hens by low-dose DFP results in an overall pattern of axonal degeneration like that produced by a threefold higher dose of DFP alone, and support the hypothesis that potentiation causes an increase in the frequency of axonal lesions in central and peripheral loci normally affected by OPIDN.

PMID: 9242229 [PubMed - indexed for MEDLINE]


Neurobiol Learn Mem 1995 Mar;63(2):116-32

Spatial working and reference memory in rats bred for autonomic sensitivity to cholinergic stimulation: acquisition, accuracy, speed, and effects of cholinergic drugs.

Bushnell PJ, Levin ED, Overstre
et DH.

Neurotoxicology Division, United States Environmental Protection Agency Research Triangle Park, NC 27711, USA.

Rat lines were selected by breeding for sensitivity to signs of autonomic stimulation (hypotherma, loss of body weight, and reduced water intake) induced by the cholinesterase inhibitor diisopropyl fluorophosphate (DFP). These lines have since been maintained for 10 generations by continued selection for hypothermic responsiveness to the muscarinic agonist oxotremorine. The sensitive rats (Flinders Sensitive Line, FSL) differ from the resistant rats (Flinders Resistant Line, FRL) both neurochemically and behaviorally, particularly in aversively motivated test situations in which response speed is assessed. This study was conducted to determine whether the selected differences in cholinergic autonomic sensitivity would be expressed as differences in cognitive ability based on choice accuracy in appetitive tasks. The working and reference memory of rats of these two strains was thus assessed using operant delayed matching-to-position/visual discrimination (DMTP/VD) and the radial-arm maze. A Long-Evans (L-E) reference group was included in the DMTP/VD study. FSL rats responded more slowly than the other rats during acquisition of both tasks, but showed no differences in response accuracy either during acquisition or during asymptotic performance of either task. In addition, challenges with muscarinic and nicotinic antagonists and agonists [scopolamine (0.06-1.0 mg/kg), pilocarpine (1.0-4.0 mg/kg), mecamylamine (1.0-10.0 mg/kg), and nicotine (0.1-0.3 mg/kg)] demonstrated predicted differences in sensitivity among the lines only on performance measures such as response latency and trial completion. Counter to prediction, the sensitivity of the FRL rats to the ability of scopolamine to reduce matching accuracy was lower than those of the L-E and FSL rats. Thus selection based upon physiological endpoints related to cholinergic autonomic homeostasis did not produce analogous differences in cognitive function in rats.

PMID: 7663885 [PubMed - indexed for MEDLINE]


Arch Toxicol 1995;69(10):705-11

Triphenylphosphite neuropathy in hens.

Fioroni F, Moretto A, Lotti M.

Istituto di Medicina del Lavoro, Universita degli Studi di Padova, Italy.

Single doses of triphenyl phosphite (TPP), a triester of trivalent phosphorus, cause ataxia and paralysis in hens. Characteristics of neurotoxicity were described as somewhat different from organophosphate induced delayed polyneuropathy (OPIDP), which is caused by triesters of pentavalent phosphorus. The onset of TPP neuropathy was reported to occur earlier than that of OPIDP (5-10 versus 7-14 days after dosing, respectively), and chromatolysis, neuronal necrosis and lesions in certain areas of the brain were found in TPP neuropathy only. Pretreatment with phenylmethanesulfonyl fluoride (PMSF) protects from OPIDP, but it either partially protected from effects of low doses or exacerbated those of higher doses of TPP. In order to account for these differences with OPIDP, it was suggested that TPP neuropathy results from the combination of two independent mechanisms of toxicity: typical OPIDP due to inhibition of neuropathy target esterase (NTE) plus a second neurotoxicity related with other target(s). We explored TPP neuropathy in the hen with attention to the phenomena of promotion and protection which are both caused by PMSF when given in combination with typical neuropathic OPs. When PMSF is given before neuropathic OPs it protects from OPIDP; when given afterwards it exaggerates OPIDP. The former effect is due to interactions with NTE, the latter to interactions with an unknown site. The time course of NTE reappearance after TPP (60 or 90 mg/kg i.v.) inhibition showed a longer half-life when compared to that after PMSF (30 mg/kg s.c.) (10-15 versus 4-6 days, respectively). The clinical signs of TPP neuropathy (60 or 90 mg/kg i.v.) were similar to those observed in OPIDP, appeared 7-12 days after treatment, correlated with more than 70% NTE inhibition/aging and were preceded by a reduction of retrograde axonal transport in sciatic nerve of hens. TPP (60 mg/kg i.v.) neuropathy was promoted by PMSF (120 mg/kg s.c.) given up to 12 days afterwards and was partially protected by PMSF (10-120 mg/kg s.c.) when given 24 h before TPP (60 or 90 mg/kg i.v.). The previously reported early onset of TPP neuropathy might be related to the higher dose used in those experiments and to the resulting more severe neuropathy. The lack of full protection might be explained by the slow kinetics of TPP, which would cause substantial NTE inhibition when PMSF effects on NTE had subsided. Since PMSF also affects the promotion site when given before initiation of neuropathy, the resulting neuropathy would then be due to both protection from and promotion of TPP effects by PMSF. No promotion by PMSF (120 mg/kg s.c.) was observed in TPP neuropathy (90 mg/kg i.v.) partially protected by PMSF (10-30 mg/kg s.c.). This might also be explained by the concurrent effects on NTE and on the promotion site obtained with PMSF pretreatment. We conclude that TPP neuropathy in the hen is likely to be the same as typical OPIDP. The unusual effects of combined treatment to hens with TPP and PMSF are explained by the prolonged pharmacokinetics of TPP and by the dual effect of PMSF i.e. protection from and promotion of OPIDP.

PMID: 8572929 [PubMed - indexed for MEDLINE]


Toxicol Lett 1995 Oct;80(1-3):115-21

Selective promotion by phenylmethanesulfonyl fluoride of peripheral and spinal cord neuropathies initiated by diisopropyl phosphorofluoridate in the hen.

Peraica M, Moretto A, Lotti M.

Universita degli Studi di Padova, Istituto di Medicina del Lavoro, Italy.

This paper reports studies in hens showing that diisopropyl phosphorofluoridate (DFP) neuropathy is promoted by PMSF when initiated either in central (spinal cord) or peripheral nervous system. Moreover, the critical site for promotion is in peripheral nerve axons rather than in their cell bodies. Selective promotion in peripheral nerves was achieved by giving PMSF into sciatic artery monolaterally (7 mg/kg) to birds where neuropathy was initiated by DFP, either systematically (0.3 mg/kg s.c.) or intra-arterially (0.04 mg/kg in the same artery). Birds developed monolateral neuropathy in the leg where PMSF was delivered. Promotion of spinal cord neuropathy was achieved by giving PMSF (120 mg/kg s.c.) to birds where neuropathy was initiated selectively in spinal cord. This was obtained by protecting peripheral axons with intra-arterial bilateral injections of PMSF (0.55 x 2 mg/kg) followed by DFP (0.3, 0.4 or 0.7 mg/kg s.c.). The resulting syndrome was characterized by spastic ataxia.

PMID: 7482578 [PubMed - indexed for MEDLINE]


J Pharmacol Exp Ther 1993 Aug;266(2):1007-17

Behavioral and neurochemical effects of acute chlorpyrifos in rats: tolerance to prolonged inhibition of cholinesterase.

Bushnell PJ, Pope CN, Padilla S.

Neurotoxicology Division, United States Environmental Protection Agency, Research Triangle Park, North Carolina.

The preponderance of studies of tolerance to organophosphate (OP) cholinesterase (ChE) inhibitors indicates that functional recovery accompanies neurochemical compensations for the inhibited enzyme. Contrary to prediction, rats dosed with the OP diisopropylfluorophosphate (DFP) showed progressive and persistent impairment of cognitive and motor function over a 3-week period of daily exposure, despite neurochemical and pharmacological evidence of tolerance to its inhibition of ChE. To determine whether these functional effects of DFP resulted from inhibition of ChE and downregulation of muscarinic cholinergic receptors, rats were dosed with chlorpyrifos (CPF), an OP pesticide which inhibits blood and brain ChE of rats for weeks after a single injection. Long-Evans rats were trained to perform an appetitive test of memory and motor function and were then injected s.c. with 0, 60, 125 or 250 mg/kg of CPF in peanut oil and tested 5 days/week for 7 weeks. Unconditioned behavior was also rated for signs of cholinergic toxicity. CPF inhibited ChE activity in whole blood in a dose-related manner for more than 53 days. The degree and time course of ChE inhibition in blood and brain and the downregulation of muscarinic receptors in brain after 125 mg/kg of CPF closely paralleled the previously reported effects of 25 daily injections of 0.2 mg/kg of DFP. In addition, CPF-treated rats were subsensitive to oxotremorine-induced hypothermia for at least 32 days after CPF. However, functional deficits (in working memory and motor function) appeared within 2 days after injection of CPF and recovered within 3 weeks, long before ChE activity and receptor density returned to control levels. Thus, the effects of CPF were neither progressive nor as persistent as those seen during daily DFP injections. This difference suggests that the DFP-induced behavioral changes observed previously cannot be attributed entirely to its effects on ChE activity and changes in [3H]quinuclidinyl benzilate binding.

PMID: 7689099 [PubMed - indexed for MEDLINE]


Biochem Pharmacol 1993 Jan 7;45(1):131-5

Organophosphate polyneuropathy in chicks.

Peraica M, Capodicasa E, Moretto A, Lotti M.

Universita delgi Studi di Padova, Istituto di Medicina del Lavoro, Italy.

Young animals are resistant to organophosphate-induced delayed neuropathy (OPIDP), although biochemical changes on Neuropathy Target Esterase (NTE) caused by neuropathic organophosphorus esters (OP) are similar to those observed in the sensitive hen. We report here that the resistance of chicks to single doses of neuropathic OPs is not absolute because ataxia was produced in 40-day-old chicks by 2,2-dichlorovinyl dibutyl phosphate (DBDCVP, 5.0 or 10.0 mg/kg s.c.) and by diisopropyl phosphorofluoridate (DFP, 2.0 mg/kg s.c.). However, the clinical picture was different from that usually seen in hens; spasticity and complete recovery being the main features. alpha-Tolyl sulphonyl fluoride (PMSF, 300 mg/kg s.c.) promoted both DBDCVP neuropathy (5.0 or 10.0 mg/kg s.c.) and non-neuropathic doses of DFP (1.5 mg/kg s.c.) or DBDCVP (1.0 mg/kg s.c.). The lowest promoting dose of PMSF given 24 hr after 1.5 mg/kg of DFP was 30 mg/kg. Higher doses had a more severe effect but no further increase of OPIDP severity was obtained with doses ranging from 90 to 300 mg/kg. PMSF (30 mg/kg) protected 40-day-old chicks from subsequent doses of neuropathic OPs even when a promoting dose of PMSF followed. At 60 days of age, chicks' resistance to OPIDP decreased because lower doses of neuropathic OPs became effective and, similarly to hens, PMSF did not fully protect from subsequent promotion. In 40-day-old chicks the threshold of NTE inhibition for OPIDP development was 95-97% (DBDCVP 5.0 mg/kg). When promotion followed initiation, the minimal effective inhibition of NTE for initiation by neuropathic OPs was about 90%. In 36-day-old chicks, PMSF (300 mg/kg) promoted OPIDP when given up to 5 days after DFP (1.5 mg/kg) when residual NTE inhibition in brain and sciatic nerve was about 40%. We conclude that chicks' resistance to OPIDP might reflect either a less effective initiation by phosphorylated NTE or a more efficient repair mechanism or both, and also that promotion is likely to involve a target other than NTE.

PMID: 8381002 [PubMed - indexed for MEDLINE]

From Dart Special at Toxnet

Journal of Occupational Medicine and Toxicology 1993;2(4):383-97

A review article on placental transfer of pesticides.

Salama AK, Bakry NM, Abou-Donia MB

Department of Pesticide Chemistry, University of Alexandria, Egypt.

Lipid-soluble pesticides generally have no problem in reaching the fetus. Since these lipid-soluble chemicals are expected to diffuse quickly, their final accumulation must be decided by partitioning against the blood of the mother. On the other hand, polar chemicals or polar metabolites of pesticides are expected to reach the fetus slowly, but once there they have a very slow rate of elimination. The possible mechanisms by which substances cross the placenta and reach the fetus may be considered under four main headings: simple diffusion, facilitated diffusion, active transport, and special processes. Many organochlorine pesticides such as DDT, DDE, DDD, HCH, PCBS, aldrin, dieldrin, and heptachlor epoxide have been found in newborn infants in America, Canada, Israel, India, Western Solvakia and Japan. Also, methoxychlor, nitrofen, kepone, and mirex were found to cross the placenta in laboratory animals. Similarly, organophosphorous pesticides, e.g., parathion, methyl parathion, methyl paraoxon, DFP, dichlorovos, diazinon, apholate, malathion, TEPA, imidan, bromophos, trichlorfon, phosfolan, mephosfolan, methamidophos and acephate have been transferred to the fetus via placenta. In addition to these pesticides, carbaryl, methiocarb, mexacarbate, 2,4-D,2,4,5-T, paraquat, diquat, and nicotine were also transplacentally passed to the fetus.


Neurotoxicology 1992 Summer;13(2):355-64

Phenylmethylsulfonyl fluoride alters sensitivity to organophosphorus-induced delayed neurotoxicity in developing animals.

Pope CN, Chapman ML, Tanaka D Jr, Padilla S.

School of Pharmacy, Northeast Louisiana University, Monroe 71209.

The serine/cysteine hydrolase inhibitor phenylmethylsulfonyl fluoride (PMSF) markedly intensifies the clinical expression of organophosphorus-induced delayed neurotoxicity (OPIDN) in adult chickens when administered after organophosphate exposure. In this study, we have examined the ability of PMSF post-treatment to affect sensitivity to OPIDN in developing animals at ages normally showing resistance. Chickens (35, 49 or 70 days of age) were treated with diisopropylphosphorofluoridate (DFP, 2 mg/kg, sc) and then treated four hours later with PMSF (90 mg/kg, sc) or vehicle only and examined for clinical signs of ataxia and incoordination. Chickens treated with DFP alone showed a marked age-related increase in the severity of motor deficits. Birds treated with DFP followed by PMSF showed more extensive clinical deficits relative to those treated with DFP only, but relatively similar degrees of motor dysfunction among the age groups. Cervical spinal cord samples processed by the Fink-Heimer degeneration method indicated that PMSF post-treatment induced more extensive axonal degeneration in all age groups relative to treatment with DFP only. As the DFP treatment alone caused greater than or equal to 90% inhibition of neurotoxic esterase activity (NTE, the putative molecular target site for OPIDN), interaction with NTE by PMSF does not appear to be involved in potentiation. We hypothesize that PMSF potentiates OPIDN through impairment of a physiological process which normally imparts resistance to young animals and which regresses during development.

PMID: 1436755 [PubMed - indexed for MEDLINE]


J Pharmacol Exp Ther 1991 Feb;256(2):741-50

Behavioral and neurochemical changes in rats dosed repeatedly with diisopropylfluorophosphate.

Bushnell PJ, Padilla SS, Ward T, Pope CN, Olszyk VB.

Neurotoxicology Division, United States Environmental Protection Agency, Research Triangle Park, North Carolina.

Behavioral effects of organophosphates (OPs) typically decrease with repeated exposure, despite persistence of OP-induced inhibition of acetylcholinesterase (AChE) and downregulation of muscarinic acetylcholine (ACh) receptors. To characterize this tolerance phenomenon, rats were trained to perform an appetitive operant task which allowed daily quantification of working memory (accuracy of delayed matching-to-position), reference memory (accuracy of visual discrimination) and motor function (choice response latencies and inter-response times during delay). Daily s.c. injections of 0.2 mg/kg of diisopropylfluorophosphate (DFP) caused no visible cholinergic signs, did not affect body weight or visual discrimination, but progressively impaired matching accuracy and lengthened response latencies and interresponse times. These effects recovered in seven of eight treated rats after termination of DFP treatment. Resumption of daily DFP at 0.1 mg/kg caused smaller impairments of both matching accuracy and response latency. After 21 injections of 0.2 mg/kg/day of DFP, rats were subsensitive to the hypothermia induced by acute oxotremorine (0.2 mg/kg i.p.), as expected after OP-induced downregulation of muscarinic ACh receptors. Evidence for supersensitivity to scopolamine (0.03 and 0.056 mg/kg i.p.) in DFP-treated rats was mixed, with additive effects predominating on both the cognitive and motor aspects of the task. After 18 days of 0.1 mg/kg of DFP, AChE was inhibited 50 to 75% and muscarinic ACh receptor density was reduced 15 to 20% in hippocampus and frontal cortex. Progressive declines in AChE activity in hippocampus and frontal cortex across 15 daily doses with DFP at 0.1 and 0.2 mg/kg were observed in other rats; quinuclidinyl benzilate binding was significantly reduced in hippocampus after 15 doses at both levels of DFP. These results indicate that animals showing a definitive sign of tolerance to OP administration (subsensitivity to a cholinergic agonist) were also functionally impaired on both the mnemonic and motoric demands of a working memory task. The nature of this impairment suggests further that it results from compensatory changes in the central nervous system, e.g., muscarinic receptor downregulation, considered to produce "tolerance" to OPs in exposed animals.

PMID: 1994004 [PubMed - indexed for MEDLINE]


J Toxicol Environ Health 1990 Dec;31(4):261-73

Potentiation of organophosphorus-induced delayed neurotoxicity by phenylmethylsulfonyl fluoride.

Pope CN, Padilla S.

School of Pharmacy, Northeast Louisiana University, Monroe 71209.

It is well known that pretreatment with the serine esterase inhibitor phenylmethylsulfonyl fluoride (PMSF) can protect experimental animals from organophosphorus-induced delayed neurotoxicity (OPIDN), presumably by blocking the active site of neurotoxic esterase (NTE) such that binding and "aging" of the neuropathic OP is thwarted. We report here that while PMSF (60 mg/kg, sc) given 4 h before the neuropathic organophosphate (OP) mipafox (50 mg/kg, im) completely prevented the clinical expression of OPIDN in hens, the identical PMSF treatment markedly amplified the delayed neurotoxicity (relative to hens treated with OP only) if administered 4 h after mipafox (5 or 50 mg/kg, im). Moreover, in a separate experiment using diisopropylphosphorofluoridate (DFP) as the neurotoxicant in place of mipafox, posttreatment with PMSF 4 h after DFP (0.5 mg/kg) also accentuated the severity of ataxia. These data indicate that PMSF only protects against OPIDN if given prior to exposure to the neurotoxicant; treatment with PMSF after OP exposure critically exacerbates the delayed neurotoxicity from exposure to organophosphorus compounds.

PMID: 2254952 [PubMed - indexed for MEDLINE]

From Dart Special at Toxnet

FASEB J 1990;4(7):A2051

Yolk sac membrane targets of the organophosphorus insecticides.

Suntornwat O, Kitos P

Dept. of Biochem., Univ. of Kansas, Lawrence, KS.

Many organophosphorus (OP) insecticides are teratogenic to vertebrates, acting on acetylcholineesterase, kynurenine formamidase, and possibly other serine esterases. We sought to identify their protein targets in the yolk sac membrane (YSM) and to correllate their OP binding with developmental changes. OP compounds with different teratogenic potentials were administered to chicken eggs at day 4 and the eggs were incubated to day 10 when the YSMs were removed and homogenized. Cell free extracts were prepared and incubated with [3H]diisopropyl fluorophosphate. The reaction mixture was then boiled with sodium dodecylsulfate (SDS) and beta-mercaptoethanol and the proteins in it were resolved by SDS PAGE. The gels were fixed, stained and either autofluorographed or sliced and the amount of radioactivity in each slice was determined. There were 3 major DFP-binding proteins in the YSM, #1 94 KD; #2 83 KD; #3 72 KD, accounting for 8, 4 & 88% of the DFP binding, respectively. Pretreatment of the embryos with 1.6 umole/egg of EPN, a mildly teratogenic insecticide, completely suppressed DFP binding to peak 3 but suppressed binding to peaks 1 & 2 only 50%. An equal amount of diazinon, a highly teratogenic insecticide, suppressed binding to peak 3 by 50% and to peaks 1 & 2 by 80%. These and other findings suggest that OP binding to peak 3 does not have teratogenic implication but binding to peaks 1 & 2 does.


Toxicol Appl Pharmacol 1987 Mar 30;88(1):87-96

Central-peripheral delayed neuropathy caused by diisopropyl phosphorofluoridate (DFP): segregation of peripheral nerve and spinal cord effects using biochemical, clinical, and morphological criteria.

Lotti M, Caroldi S, Moretto A, Johnson MK, Fish CJ, Gopinath C, Roberts NL.

Systemic injection of diisopropyl phosphorofluoridate (DFP; 1 mg/kg, sc) causes delayed neuropathy in hens. This effect is associated with a high level of organophosphorylation of neuropathy target esterase (NTE) followed by an intramolecular rearrangement called "aging." Phenylmethanesulfonyl fluoride (PMSF) also attacks the active center of NTE but "aging" cannot occur. This compound does not cause neuropathy and protects against a subsequent challenge systemic dose of DFP. Intraarterial injection of DFP (0.185 mg/kg) into only one leg of hens caused a high NTE inhibition (greater than 80%) in the sciatic nerve of the injected leg, but not in other parts of the nervous system (37% average). A unilateral neuropathy with typical histopathological lesions developed in the injected leg. PMSF (0.55 mg/kg) injected into each sciatic artery caused 47% inhibition of sciatic nerve NTE but only 17-22% inhibition of NTE elsewhere; it did not produce clinical or histopathological lesions. When these hens were challenged with DFP (1 mg/kg, sc), high inhibition of residual-free NTE (greater than 85%) occurred throughout the nervous system and clinical signs of a syndrome different from the classical delayed neuropathy developed: this spinal cord type of ataxia was associated with histopathological lesions in the spinal cord but not in peripheral nerve. PMSF (1 mg/kg) injected into only one sciatic artery caused selective protective inhibition of sciatic nerve NTE of that leg. After systemic challenge by DFP, clinical effects expressed were a combination of spinal cord ataxia plus unilateral peripheral neuropathy. The challenge dose of DFP (1 mg/kg, sc) was insufficient to produce clear histopathological lesions in unprotected peripheral nerves although spinal lesions were found in these hens. Thus clinical evaluation of the peripheral nervous system by means of walking tests and a simple test of "leg retraction" reflexes was more sensitive and specific in diagnosis of peripheral neuropathy than was the histopathology.

PMID: 3564033 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=766733&dopt=Abstract \

Arch Toxicol 1975 Dec 18;34(4):259-88

Organophosphorus esters causing delayed neurotoxic effects: mechanism of action and structure activity studies.

Johnson MK.

Evidence is reviewed that the initial biochemical event leading to delayed neurotoxicity is phosphorylation of the active site of a specific enzyme called Neurotoxic Esterase. This is followed by a bondcleavage (? hydrolytic) leading to formation of a mono-substituted phosphoric acid residue on the protein. The mechanism by which some phosphinates protect hens against neurotoxic compounds is explained. Screening Assay. Assay of effects of compounds on Neurotoxic Esterase activity of hen brain in vitro and in vivo provides a quick biochemical screen to supplement the 3-week clinical test. This test provides an estimate of safety margin for compounds which give negative results in the clinical test and are currently used as pesticides, plasticisers, etc. Simplified assay procedures are being developed. Structure/Activity Studies. Data is now available for the biochemical and neurotoxic activity of many compounds. This provides a basis for structure/activity predictions; neurotoxicity data published since 1930 has been assessed in this light.

Publication Types:

PMID: 766733 [PubMed - indexed for MEDLINE]


Biochem J 1970 Dec;120(3):523-31

Organophosphorus and other inhibitors of brain 'neurotoxic esterase' and the development of delayed neurotoxicity in hens.

Johnson MK.

PMID: 5515416 [PubMed - indexed for MEDLINE]

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