http://pubs.acs.org/cgi-bin/abstract.cgi/crtoec/2006/19/i02/abs/tx050342o.html
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.
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12591136&dopt=Abstract
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]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11958515&dopt=Abstract
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]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11754869&dopt=Abstract
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]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11570692&dopt=Abstract
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]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11310568&dopt=Abstract
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]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11495547&dopt=Abstract
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.
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10373406&dopt=Abstract
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]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9242229&dopt=Abstract
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]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7663885&dopt=Abstract
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, Overstreet
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]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8572929&dopt=Abstract
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]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7482578&dopt=Abstract
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]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7689099&dopt=Abstract
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]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8381002&dopt=Abstract
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.
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1436755&dopt=Abstract
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]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1994004&dopt=Abstract
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]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=2254952&dopt=Abstract
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.
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3564033&dopt=Abstract
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: Review
PMID: 766733
[PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=5515416&dopt=Abstract
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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