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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Neurotoxicity of organophosphorus insecticides leptophos and EPN a
a
A.H. El‐Sebae , S.A. Soliman , M. Abo a
Elamayem & Nabila S. Ahmed
a
a
Plant Protection Department, Faculty of Agriculture , Alexandria University , Alexandria, Egypt Published online: 21 Nov 2008.
To cite this article: A.H. El‐Sebae , S.A. Soliman , M. Abo Elamayem & Nabila S. Ahmed (1977) Neurotoxicity of organophosphorus insecticides leptophos and EPN, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 12:4, 269-288, DOI: 10.1080/03601237709372071 To link to this article: http://dx.doi.org/10.1080/03601237709372071
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our
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J. ENVIRON. SCI. HEALTH, B12(4), 269-288 (1977)
NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES LEPTOPHOS AND EPN. KEY WORLDS: Delayed n e u r o t o x i c i t y , neurotoxic Downloaded by [ECU Libraries] at 01:15 21 April 2015
e s t e r a s e and mono amine oxidase i n h i b i t o r s , Leptophos, BPN. A.H. El-Sebae, S.A. Soliman, M. Abo Elamayem and Nabila S. Ahmed Plant P r o t e c t i o n Department, Faculty of Agriculture, Alexandria U n i v e r s i t y , Alexandria, Egypt. ABSTRACT Phosfolan, c h l o r p y r i f o s , and s t i r o p h o s when applied t o white mice a t s u b l e t h a l doses d i d not induce any delayed neurotoxic e f f e c t .
On t h e other hand, Leptophos and EPN
when administered o r a l l y a t s u b l e t h a l o r l e t h a l l e v e l s c l e a r l y produced, a delayed neurotoxic a t a x i a i n t r e a t e d mice.
The
f i v e t e s t e d organophosphorus i n s e c t i c i d e s were compared f o r their ability to inhibit cholinesterase, neurotoxic esterase and monoamine oxidase. I50 values were estimated for each case.
The results revealed that a l l five compounds were
inhibitors of cholinesterase, but only Leptophos andEPNwere shown to be potent inhibitors for both neurotoxic esterase
269 Copyright © 1977 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher..
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and monoamine oxidase i n the mouse brain.
Additional p a r t i c u l a r
properties of both Leptophos and EPN were found i n t h e i r a b i l i t y t o cause delayed neurotoxic ataxia i n chickens and sheep fed once on sublethal doses of these compounds. I t i s believed t h a t t h e phosphonate ester configuration of EPH and Leptophos has a specific mode of toxic action
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which i s mainly located a t the c e n t r a l nervous system. I t i s also postulated t h a t these delayed neurotoxic agents might i n h i b i t postganglionic sympathetic neurons, thus r e s u l t i n g i n chronic p a r a l y t i c e f f e c t s .
INTRODUCTION In addition t o t h e widely known toxic effect of organophosphorus compounds as anticholinetit erases, some OP-esters a r e characterized by exhibiting a specific delayed neurotoxicity t o vertebrates.
The symptoms are
shown as f l a c c i d paralysis especially of t h e legs i n man and b i r d s , o r hind limbs of c a t t l e and other quadrupeds. These symptoms appear only two weeks or more a f t e r poisoning and therefore i t i s called delayed neurotoxicity. The precise biochemical action t h a t causes t h e damage i s s t i l l unknown. The phenomenon was called demylination because i t i s associated with lesions i n the mylein sheath which cover nerve f i b e r s and i s e s s e n t i a l t o nerve t r a n s mission . Later work showed however, t h a t the neuro axon i t s e l f was primarily affected and t h a t t h e damage t o myelin was only secondary.
NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES
271
Recently i t was proved that the target site in the central nervous system i s a specific protein called neurotoxic esterase which i s present in the brain and p
spinal cord of suceptible species . The f i r s t report of delayed neurotoxicity to humans occurred in 1930's in the midwestern and south-western
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United States during prohibition days as the result of drinking alcoholic Jamaica ginger adulterated by the etiological agent tri-orthocresyl phosphate (TOCP). I t resulted in an outbreak of cases of so called "ginger" or "Jake leg" paralysis.
The second incident happened
in Morosco in the 1959's, when several thousands of people developed the typical paralysis following the use of an imported cooking o i l which was contaminated with motor oil containing TOCP as a combustion deposit modifier . In 1953 cases of neuropathy were reported in three workers enganged i n laboratory production of a prospective organophosphate pesticide, mipafox (N,H-diisopropyl phosphoro-2,3-diamidofluoridate) in united Kingdom. In 1971 a mysterious epidemic of paralysis struck several hundred water buffaloes i n Egypt and eventually resulted in the death of 1200 to 1300 animals.
Evidence
strongly pointed to Leptophos (0-(4-bromo-2,5-dichlorophenyl) 0-methyl phenylphosphonothioate) as responsible for this neurotoxic syndrome. A more curious event took place as six people were discovered to develop symptoms of neurotoxic poisoning, and traces of Leptophos were found in their tissues*.
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EL-SEBAE ET AL.
Further laboratory studies proved that Leptophos i s neurotoxic to rabbits. Ataxia attributable to degeneration of nervous tissues was confirmed by histopathological lesions recorded in white matter of the cerebellum. Delayed neurotoxicity was also shown in chickens at a dose 25 times less than the lethal dose. This effect
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was proved histopathologically in the sciatic nerve, the spinal cord and the brain of chickens ' . The present investigation i s devoted to investigate the actual site and mechanism of toxic action of some phosphorokioate esters which might show such delayed neurotoxicity. MATERIALS AND METHODS
Insecticides Leptophos 30% E.C.O-CA-b 0-methyl phenylphosphonothioate) was supplied by Velsicol Corp.; EPN 30% (O-ethyl O-(p-nitrophenyl) phenyl phosphonothioate) was supplied by Hissan Co.; Chlorpyriphos (Dursban) 40% E.G. (0,0-diethyl 0-(3,5,6-trichloro-2-pyridyl) phosphorothioate) was supplied by Dow Chem. Co.; Stirophos (gardona) 70% S.C. (2-chloro-l-(2,4,5-trichloro-phenyl) vinyl dimethyl phosphate) from Shell International Chem. Co.; and Phosfolan (Cyolane) 25% E.C. (P,?-diethyl cyclic ethylene ester of phosphonodithioimidocarbonic acid) was supplied by American Cyanamid Co* Delayed Heurotoxicity by Leptophos i n Chickenst Female chickens s i x months old of local breed
NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES
273
(Alexandrian) each weighing approximately 1.5 kg were placed in open pens.
Each bird received a single dose
of 30% E.C. Leptophos. oral intubation. food and water.
Each dose was administered with
The birds were given free access to For each dose at least three birds were
used as replicates.
The dose killing 50% of the birds
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within 24 hours was recorded as IJ^CQ*
^ û e surviving
birds were observed every 2 days for 60 days or until they died. An adequate untreated group of chickens were included in every t e s t .
Symptoms were recorded and
photographed. Delayed Heurotoxicity by Leptophos in Sheep: Two crossbred lambs one year old, each weighing approximately 30 kg were used in the preliminary t e s t . One was kept as an untreated control and the second was fed a daily ration consisting of 5 kg of freshly cut clover (Trifolium alexandrinum).
The daily ration of clover was
sprayed with 1.9 ml of 30% E.C. Leptophos
every other day
in order to imitate the natural field conditions where this insecticide i s applied at the rate of 2.25 l i t r e s of formulated material per feddan (1.05 acre).
The
total weight of one feddan ready to cut clover i s equal to 6000 kg.
Accordingly, the 5 kg clover were sprayed
with 1.9 ml of the formulated Leptophos 30% E.C.
After
two weeks both animals were fed with normal unsprayed clover, and were kept under observation during the next fifteen weeks. The symptoms were recorded and photographed.
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EL-SEBAE ET AL.
Neurotoxicity Tests in White Mice: Adult white mice weighing from 10-18 gm were used in this study. These animals were taken from a colony raised at the High Institute of Public Health, University of Alexandria.
The corn oil solutions of formulated
insecticides were administered orally to mice, in volumes Downloaded by [ECU Libraries] at 01:15 21 April 2015
not exceeding 0.2 ml to avoid régurgitation using a plastic tuberculin syringe. The following tests were included on white mice: a- Delayed neurotoxicity effects: Delayed neurotoxicity was detected for either Leptophos or EPN by applying sublethal doses to white mice* The treated mice were kept under'observation for 15 days or t i l l they died. Any symptoms of ataxia or delayed paralysis were recorded and photographed. b- In vivo and in vitro Mice-Brain AChB Inhibition: The I 5 0 values of formulated leptophos, EPN, chlorpyriphos, phosfolan, and stirophos to brain AChE were determined both in vivo and in vitro as described previously ously . c- In vitro Inhibition of the Mice-Brain Neurotoxic Esterase: The in vitro I g n values of the tested formulated insecticides to mice brain neurotoxic esterase were determined |J| substrate.
.
Phenyl valerate was used as a
DD7P (30 uM) was used for the inhibition of
esterases other than neurotoxic esterase.
NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES
275
d
~ ¿ a v i t r o Inhibition of Monoamine Oxidase i n White Mice-Brain: Monoamine oxidase (MAO) enzyme preparation was
obtained from brains of f i v e white mice animals. The 11 12 n J enzyme purification • , and assay by using benzylamine
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as a substrate was carried out. The r a t e of benzaldehyde l i b e r a t i o n was measured spectrophotometrically (250 nm). Time of incubation of the enzyme with inhibitors was 15 minutes, and that between treated enzyme and benzylamine was 10 minutes. RESULTS AND DISCUSSION 1- Delayed Neurotoxicity of Leptophos i n Chickens; Three separate experiements with different doses proved that the 24 hours LD,-0 of 30$ E.C. Leptophos was 1400 mg/kg. The survivore began to show loss of self control, i n a b i l i t y to move and weakness of the wings as shown i n figures 1 and 2. Ataxia developed a f t e r 3-6 days and i t was not r e versible.
Comparing the LD50 value of the formulated 30%
E.C. Leptophos with t h e corresponding value f o r t h e technical material
, i t i s clear that the formulated material i s
more toxic and hazardous than the technical material. Therefore, i t i s recommended that the formulated material i s more r e l i a b l e i n predicting the real hazards t o the environment.
EPN i s early known t o be a potent delayed
neurotoxic compound t o chickens
. On the other hand,
phosfolan, stirophos and chlorpyriphos proved not t o be of any delayed neurotoxicity.
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EL-SEBAE ET
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vM
w
f V*'.*' ^
', ' ,.*>=**8!,j
Figure 1- Ataxia in chickens treated with aublethal dose of Leptophos.
Eigure 2- Advanced inability in chickens treated with sublethal dose of Leptophos.
AL.
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NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES
Ï s
Figure 3- Locomotor Ataxia in hind lings in sheep treated with sublethal dose of Leptophos,
Figure 4- Advanced inability of sheep treated with sublethal dose of Leptophos.
277
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EL-SEBAE ET AL.
2- Delayed Neurotoxicity of Leptophoa to Sheep; The subacute feeding study on Leptophos-sprayed clover to sheep resulted in irreversible symptoms of ataxia to the hind limbs after 60 days as shown in figures 3f and 4. ThiB typical delayed neurotoxicity caused by Leptophos to sheep adds a new example of farm animals which i s susDownloaded by [ECU Libraries] at 01:15 21 April 2015
ceptible for this delayed neurotoxic agent.
In Egypt,
i t was previously recorded that Leptophos caused delayed neurotoxicity in water buffaloes
. Cattle and Jamb are
considered as susceptible to demylination by TOCP as are the chickens and man17. 3- Delayed Heurotoxicity in White Mice: Several separate experiments gave evidence that Leptophos at sublethal doses was able to exert ataxia in the hind legs of white mice but at shorter time intervals than those required in the previous cases of sheep and chickens. Leptophos in a single sublethal dose (30 mg/kg) induced irreversible ataxia in the hind legs of white mice after 3 days.
(Figure 5).
EPN also in a single sublethal
dose (20 mg/kg) exerted irreversible ataxia in the hind legs of white mice but after 29 days (figure 6 ) . The two recorded cases of ataxia caused by Leptophos to sheep and white mice are original findings which were not reported before in the literature. 4- Comparative Inhibitory Effect Against Three Brain Enzymes; The five organophosphorus compounds tested were
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NEUROTOXICITY OF ORGAHOPHOSPHORUS INSECTICIDES
279
Figure 5- Ataxia in white mice treated with sublethal dose of Leptophos
Figure 6- Ataxia i n white mice treated with sublethal dose of EPN.
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Table 1 In-Vitro I n h i b i t i o n
of Mice Brain Enzymes by
D i f f e r e n t OP-Insecticides
In-vitro p 1,-Q
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Common name
Acetyl cholinesterase
of brain enzymes Neurotoxic esterase
Monoamine oxidase
Leptophos
4.658
6.895
6.12
EPN
5.520
5.347
4.10
Chlorpyrifos
5.602
NS*
4.00
Phosfolan
5.420
NS*
4.00
Stirophos
4.602
NS 1
4.00
not significant. compared for their inhibitory effect to acetyl cholinesterase (AChE), neurotoxic esterase (NIE), and monoamine oxidase (MAO) as shown in table 1. The five OP compounds exerted different degrees of inhibition to AChE, and the most potent inhibitor was chlorpyriitos , followed by EPN, phosfolan, Leptophos, and stirophos in a descending order* On the other hand, Leptophos was almost 100 fold as potent to both NTE and MAO followed by EPN while the other three candidate compounds did not show any apprecibable inhibition of either NTE or MAO.
This clear specific affinity of
NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES
281
Leptophos or EPN for blocking NTE or MAO must be of significance and should be taken i n consideration in any interpretation of the characteristic effect of these two compounds in inducing symptoms of delayed neurotoxicity. In recent reports, in-vitro inhibition of MTE i s taken as a precise criterion in detecting the delayed neurotoxic
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agents ' .
Thus evidence began to support the impression
that acetylcholinesterase inhibition i s not the solely mode of toxic action for OP compounds especially those capable of causing delayed neurotoxicity. If the chemical structure i s considered, i t can also be deduced that the phosphonate structure in Leptophos and EPN has a positive correlation with either delayed neurotoxicity or NTE and MAO inhibition.
Chlorpyrifos , phosfolan
and stirophos did not show neither delayed neurotoxicity symptoms nor specific inhibition of NTE or MAO. The Mode of Delayed Neurotoxic Action for Some Órgano— phosphorus Compounds : The common belief was that organophosphorus compounds generally exert their acute toxicity through their ability to inhibit the cholinesterase i n the nervous system. Yet some of these OP esters are characterized by being capable of inducing irreversible chronic delayed neurotoxicity as was shown by TOCP, Mepafox, leptophos and EPN. The machanism of this chronic delayed neurotoxicity
cannot
be interpretted according to their effect as ChE inhibitors. I t was interesting that in the rat liver, isocarbonazid amidase was shown to be a more sensitive indicator of
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exposure to EPN intoxication than serum cholinesterase 18 inhibition . This assures the fact that there are other biological sites which are highly affected by OP compounds. Early detection of delayed neurotoxic agents depends upon histopathological examination showing predominantly degeneration of the fatty myelin sheath surrounding long Downloaded by [ECU Libraries] at 01:15 21 April 2015
nerve axons.
Therefore, the term demylinating agents
was used to differentiate such compounds. However, later work showed that the nerve axon itself was primarily affected and damage to myelin was only a secondary effect °. Recently, i t was observed that the chronic ataxia was accomanied by the inhibition of the brain neurotoxic esterase ' .
However, the actual role of the brain
neurotoxic esterase inhibition in exerting delayed neurotoxicity i s s t i l l vague, and the chain of events from this initial alteration of the protein to degeneration of the nerve axon i s s t i l l unknown. Besides, the function of this phosphorylated protein responsible for neurotoxic esterase activity in the brain i s s t i l l also obscure. This situation necessitates and suggests the following postulation.
The chronic ataxia and irreversible para-
lysis of the hind legs i s expected to take place due to lesion in the central nervous system which i s directly connected to the spinal cord and the sciatic nerve. Evidence i s accumulating to prove that both adrenergic and cholinergic systems are interacting at the synapases of the neuromuscular junctions.
NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES
283
It was reported that the sympathetic neurons at the brain adrenal medulla liberate acetylcholine which in turn evokes a secretion of adrenaline and noradrenaline
20
.
Recently, i t was shown that two types of cholinergic receptors-muscarinic and nicotinic- are present on adrenergic nerve terminals in sympathetic effector organs.
Clear
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direct evidence for prejunctional nicotinic receptors came from the findings that in prefused organs administration of nicotine or acetylcholine (ACh) resulted in the release of norepinephrine (NE) and in the initiation of antidromic action potentials in sympathetic nerve fibers to the organs.
Similar evidence for prejunctional muscarinic
receptors on adrenergic nerve terminals was also shown. The activation of these receptors by ACh and methacholine at low concentrations inhibited the release of (NE) evoked either by nerve stimulation or by stimulation of the neuronal nicotinic receptors, i t was also concluded that in the central nervous system, presynaptic inhibitory muscarinic receptors may well be present on dopaminergic as well as adrengergic terminals 21 . In another study i t was concluded that activation or impairment of dapaminergic transmission results in opposite effects on the release of ACh from the striatum. That mutual influence of DA and ACh neurons in the extrapyramidal centers was considered of both physiological and patho— physiological reflections22.
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AL.
Parallel to this correlation, a recent study had shown the implication of the dopaminergic activity on the dorsal and ventral locomotor activity in the r a t . I t was found that selective and extensive destruction of the nigroneostriatal DA projection, so that neostriatal DA levels were reduced to 7.5% of controls, resulted in a profound
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attenuation but not a complete blockage of the locomotor stimulant effect of D-emphetamine. Besides, the possibility that norepinepherine in the cerebellum or midbrain may mediate some of the stimulant effects of amphetamine cannot be excluded •*. All these interactions support the conclusion that the delayed neurotoxic agents should not be considered solely as anticholinesterases.
They are expected to have
a multitoxic mechanism in which their inhibitory effect will interact simultaneously with adrenergic, dopaminergic and cholinergic sites.
The released ACb during the acute
toxicity of these OP toxicants might induce and activate adrenergic and dopaminergic agents.
The concentration of
liberated dopamine, epinepherine, and other biogenic amines will also be continuously increased by the particular ability of delayed neurotoxic agents to act as MAO
inhibitors
resulting in free release of biogenic amines at the adrenergic terminals which are highly abundant in the CHS the spinal cord in mammals.
and
These free adrenogenic
transmitters are strong intact poisons.
When they diffuse
to bind to their specific receptors on the postsynaptic membrane they will increase permeability to ions, with
NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES
285
increased metabolic processes but reduced protein biosynthesis.
This reduction in protein biosynthesis and the
depletion of ions might represent an important cause of the lesions caused in the neuroaxons
'
.
The increased
neuromembrane permeability might lead to liberation of cellular hydrolases or complex changes of molecular
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architecture of membrane-bound enzymes and the interference with oxidative processes at the level of the neuronal cytoplasm and organelles as well as with energy transference and utilization mechanism. All these effects might be included as possible causes of irreversible neurotoxic effects.
This interpretation i s generally
supported by the present data showing that leptophos and EPN are characterized by being strong monoamine oxidase inhibitors as well as potent delayed neurotoxic agents. Their effect as neurotoxic esterase inhibitors might represent one of the steps in blocking the protein synthesis reactions. This postulated mechanism draws the attention to the importance of considering the adrenergic and dopaminergic neurone as the main c r i t i c a l sites of toxic action for the delayed neurotoxic agents in mamnals. The long required stepwise mechanism of such chain interactions might explain the elapsed time required to reach the delayed symptoms of those neurotoxic agents. Also the same complex mechanism leads to the irreversible lesions in the central nervous system.
The chemical
stability, persistance and high lipophiliclty of the
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two compounds Leptophos and EPîï support t h e i r ability to exist i n active form long enough to exert the delayed neurotoxic effects in the CNS of mammals and birds.
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ACKNOWLEDGEMENT
This work was p a r t i a l l y supported by a grant from the project (PR 3-545-1) financed by t h e U.S.E.P.A. Their assistance and cooperation i s highly appreciated.
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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Neurotoxicity of organophosphorus insecticides leptophos and EPN a
a
A.H. El‐Sebae , S.A. Soliman , M. Abo a
Elamayem & Nabila S. Ahmed
a
a
Plant Protection Department, Faculty of Agriculture , Alexandria University , Alexandria, Egypt Published online: 21 Nov 2008.
To cite this article: A.H. El‐Sebae , S.A. Soliman , M. Abo Elamayem & Nabila S. Ahmed (1977) Neurotoxicity of organophosphorus insecticides leptophos and EPN, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 12:4, 269-288, DOI: 10.1080/03601237709372071 To link to this article: http://dx.doi.org/10.1080/03601237709372071
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J. ENVIRON. SCI. HEALTH, B12(4), 269-288 (1977)
NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES LEPTOPHOS AND EPN. KEY WORLDS: Delayed n e u r o t o x i c i t y , neurotoxic Downloaded by [ECU Libraries] at 01:15 21 April 2015
e s t e r a s e and mono amine oxidase i n h i b i t o r s , Leptophos, BPN. A.H. El-Sebae, S.A. Soliman, M. Abo Elamayem and Nabila S. Ahmed Plant P r o t e c t i o n Department, Faculty of Agriculture, Alexandria U n i v e r s i t y , Alexandria, Egypt. ABSTRACT Phosfolan, c h l o r p y r i f o s , and s t i r o p h o s when applied t o white mice a t s u b l e t h a l doses d i d not induce any delayed neurotoxic e f f e c t .
On t h e other hand, Leptophos and EPN
when administered o r a l l y a t s u b l e t h a l o r l e t h a l l e v e l s c l e a r l y produced, a delayed neurotoxic a t a x i a i n t r e a t e d mice.
The
f i v e t e s t e d organophosphorus i n s e c t i c i d e s were compared f o r their ability to inhibit cholinesterase, neurotoxic esterase and monoamine oxidase. I50 values were estimated for each case.
The results revealed that a l l five compounds were
inhibitors of cholinesterase, but only Leptophos andEPNwere shown to be potent inhibitors for both neurotoxic esterase
269 Copyright © 1977 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher..
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and monoamine oxidase i n the mouse brain.
Additional p a r t i c u l a r
properties of both Leptophos and EPN were found i n t h e i r a b i l i t y t o cause delayed neurotoxic ataxia i n chickens and sheep fed once on sublethal doses of these compounds. I t i s believed t h a t t h e phosphonate ester configuration of EPH and Leptophos has a specific mode of toxic action
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which i s mainly located a t the c e n t r a l nervous system. I t i s also postulated t h a t these delayed neurotoxic agents might i n h i b i t postganglionic sympathetic neurons, thus r e s u l t i n g i n chronic p a r a l y t i c e f f e c t s .
INTRODUCTION In addition t o t h e widely known toxic effect of organophosphorus compounds as anticholinetit erases, some OP-esters a r e characterized by exhibiting a specific delayed neurotoxicity t o vertebrates.
The symptoms are
shown as f l a c c i d paralysis especially of t h e legs i n man and b i r d s , o r hind limbs of c a t t l e and other quadrupeds. These symptoms appear only two weeks or more a f t e r poisoning and therefore i t i s called delayed neurotoxicity. The precise biochemical action t h a t causes t h e damage i s s t i l l unknown. The phenomenon was called demylination because i t i s associated with lesions i n the mylein sheath which cover nerve f i b e r s and i s e s s e n t i a l t o nerve t r a n s mission . Later work showed however, t h a t the neuro axon i t s e l f was primarily affected and t h a t t h e damage t o myelin was only secondary.
NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES
271
Recently i t was proved that the target site in the central nervous system i s a specific protein called neurotoxic esterase which i s present in the brain and p
spinal cord of suceptible species . The f i r s t report of delayed neurotoxicity to humans occurred in 1930's in the midwestern and south-western
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United States during prohibition days as the result of drinking alcoholic Jamaica ginger adulterated by the etiological agent tri-orthocresyl phosphate (TOCP). I t resulted in an outbreak of cases of so called "ginger" or "Jake leg" paralysis.
The second incident happened
in Morosco in the 1959's, when several thousands of people developed the typical paralysis following the use of an imported cooking o i l which was contaminated with motor oil containing TOCP as a combustion deposit modifier . In 1953 cases of neuropathy were reported in three workers enganged i n laboratory production of a prospective organophosphate pesticide, mipafox (N,H-diisopropyl phosphoro-2,3-diamidofluoridate) in united Kingdom. In 1971 a mysterious epidemic of paralysis struck several hundred water buffaloes i n Egypt and eventually resulted in the death of 1200 to 1300 animals.
Evidence
strongly pointed to Leptophos (0-(4-bromo-2,5-dichlorophenyl) 0-methyl phenylphosphonothioate) as responsible for this neurotoxic syndrome. A more curious event took place as six people were discovered to develop symptoms of neurotoxic poisoning, and traces of Leptophos were found in their tissues*.
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EL-SEBAE ET AL.
Further laboratory studies proved that Leptophos i s neurotoxic to rabbits. Ataxia attributable to degeneration of nervous tissues was confirmed by histopathological lesions recorded in white matter of the cerebellum. Delayed neurotoxicity was also shown in chickens at a dose 25 times less than the lethal dose. This effect
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was proved histopathologically in the sciatic nerve, the spinal cord and the brain of chickens ' . The present investigation i s devoted to investigate the actual site and mechanism of toxic action of some phosphorokioate esters which might show such delayed neurotoxicity. MATERIALS AND METHODS
Insecticides Leptophos 30% E.C.O-CA-b 0-methyl phenylphosphonothioate) was supplied by Velsicol Corp.; EPN 30% (O-ethyl O-(p-nitrophenyl) phenyl phosphonothioate) was supplied by Hissan Co.; Chlorpyriphos (Dursban) 40% E.G. (0,0-diethyl 0-(3,5,6-trichloro-2-pyridyl) phosphorothioate) was supplied by Dow Chem. Co.; Stirophos (gardona) 70% S.C. (2-chloro-l-(2,4,5-trichloro-phenyl) vinyl dimethyl phosphate) from Shell International Chem. Co.; and Phosfolan (Cyolane) 25% E.C. (P,?-diethyl cyclic ethylene ester of phosphonodithioimidocarbonic acid) was supplied by American Cyanamid Co* Delayed Heurotoxicity by Leptophos i n Chickenst Female chickens s i x months old of local breed
NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES
273
(Alexandrian) each weighing approximately 1.5 kg were placed in open pens.
Each bird received a single dose
of 30% E.C. Leptophos. oral intubation. food and water.
Each dose was administered with
The birds were given free access to For each dose at least three birds were
used as replicates.
The dose killing 50% of the birds
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within 24 hours was recorded as IJ^CQ*
^ û e surviving
birds were observed every 2 days for 60 days or until they died. An adequate untreated group of chickens were included in every t e s t .
Symptoms were recorded and
photographed. Delayed Heurotoxicity by Leptophos in Sheep: Two crossbred lambs one year old, each weighing approximately 30 kg were used in the preliminary t e s t . One was kept as an untreated control and the second was fed a daily ration consisting of 5 kg of freshly cut clover (Trifolium alexandrinum).
The daily ration of clover was
sprayed with 1.9 ml of 30% E.C. Leptophos
every other day
in order to imitate the natural field conditions where this insecticide i s applied at the rate of 2.25 l i t r e s of formulated material per feddan (1.05 acre).
The
total weight of one feddan ready to cut clover i s equal to 6000 kg.
Accordingly, the 5 kg clover were sprayed
with 1.9 ml of the formulated Leptophos 30% E.C.
After
two weeks both animals were fed with normal unsprayed clover, and were kept under observation during the next fifteen weeks. The symptoms were recorded and photographed.
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EL-SEBAE ET AL.
Neurotoxicity Tests in White Mice: Adult white mice weighing from 10-18 gm were used in this study. These animals were taken from a colony raised at the High Institute of Public Health, University of Alexandria.
The corn oil solutions of formulated
insecticides were administered orally to mice, in volumes Downloaded by [ECU Libraries] at 01:15 21 April 2015
not exceeding 0.2 ml to avoid régurgitation using a plastic tuberculin syringe. The following tests were included on white mice: a- Delayed neurotoxicity effects: Delayed neurotoxicity was detected for either Leptophos or EPN by applying sublethal doses to white mice* The treated mice were kept under'observation for 15 days or t i l l they died. Any symptoms of ataxia or delayed paralysis were recorded and photographed. b- In vivo and in vitro Mice-Brain AChB Inhibition: The I 5 0 values of formulated leptophos, EPN, chlorpyriphos, phosfolan, and stirophos to brain AChE were determined both in vivo and in vitro as described previously ously . c- In vitro Inhibition of the Mice-Brain Neurotoxic Esterase: The in vitro I g n values of the tested formulated insecticides to mice brain neurotoxic esterase were determined |J| substrate.
.
Phenyl valerate was used as a
DD7P (30 uM) was used for the inhibition of
esterases other than neurotoxic esterase.
NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES
275
d
~ ¿ a v i t r o Inhibition of Monoamine Oxidase i n White Mice-Brain: Monoamine oxidase (MAO) enzyme preparation was
obtained from brains of f i v e white mice animals. The 11 12 n J enzyme purification • , and assay by using benzylamine
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as a substrate was carried out. The r a t e of benzaldehyde l i b e r a t i o n was measured spectrophotometrically (250 nm). Time of incubation of the enzyme with inhibitors was 15 minutes, and that between treated enzyme and benzylamine was 10 minutes. RESULTS AND DISCUSSION 1- Delayed Neurotoxicity of Leptophos i n Chickens; Three separate experiements with different doses proved that the 24 hours LD,-0 of 30$ E.C. Leptophos was 1400 mg/kg. The survivore began to show loss of self control, i n a b i l i t y to move and weakness of the wings as shown i n figures 1 and 2. Ataxia developed a f t e r 3-6 days and i t was not r e versible.
Comparing the LD50 value of the formulated 30%
E.C. Leptophos with t h e corresponding value f o r t h e technical material
, i t i s clear that the formulated material i s
more toxic and hazardous than the technical material. Therefore, i t i s recommended that the formulated material i s more r e l i a b l e i n predicting the real hazards t o the environment.
EPN i s early known t o be a potent delayed
neurotoxic compound t o chickens
. On the other hand,
phosfolan, stirophos and chlorpyriphos proved not t o be of any delayed neurotoxicity.
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EL-SEBAE ET
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vM
w
f V*'.*' ^
', ' ,.*>=**8!,j
Figure 1- Ataxia in chickens treated with aublethal dose of Leptophos.
Eigure 2- Advanced inability in chickens treated with sublethal dose of Leptophos.
AL.
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NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES
Ï s
Figure 3- Locomotor Ataxia in hind lings in sheep treated with sublethal dose of Leptophos,
Figure 4- Advanced inability of sheep treated with sublethal dose of Leptophos.
277
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EL-SEBAE ET AL.
2- Delayed Neurotoxicity of Leptophoa to Sheep; The subacute feeding study on Leptophos-sprayed clover to sheep resulted in irreversible symptoms of ataxia to the hind limbs after 60 days as shown in figures 3f and 4. ThiB typical delayed neurotoxicity caused by Leptophos to sheep adds a new example of farm animals which i s susDownloaded by [ECU Libraries] at 01:15 21 April 2015
ceptible for this delayed neurotoxic agent.
In Egypt,
i t was previously recorded that Leptophos caused delayed neurotoxicity in water buffaloes
. Cattle and Jamb are
considered as susceptible to demylination by TOCP as are the chickens and man17. 3- Delayed Heurotoxicity in White Mice: Several separate experiments gave evidence that Leptophos at sublethal doses was able to exert ataxia in the hind legs of white mice but at shorter time intervals than those required in the previous cases of sheep and chickens. Leptophos in a single sublethal dose (30 mg/kg) induced irreversible ataxia in the hind legs of white mice after 3 days.
(Figure 5).
EPN also in a single sublethal
dose (20 mg/kg) exerted irreversible ataxia in the hind legs of white mice but after 29 days (figure 6 ) . The two recorded cases of ataxia caused by Leptophos to sheep and white mice are original findings which were not reported before in the literature. 4- Comparative Inhibitory Effect Against Three Brain Enzymes; The five organophosphorus compounds tested were
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NEUROTOXICITY OF ORGAHOPHOSPHORUS INSECTICIDES
279
Figure 5- Ataxia in white mice treated with sublethal dose of Leptophos
Figure 6- Ataxia i n white mice treated with sublethal dose of EPN.
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EL-SEBAE ET AL.
Table 1 In-Vitro I n h i b i t i o n
of Mice Brain Enzymes by
D i f f e r e n t OP-Insecticides
In-vitro p 1,-Q
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Common name
Acetyl cholinesterase
of brain enzymes Neurotoxic esterase
Monoamine oxidase
Leptophos
4.658
6.895
6.12
EPN
5.520
5.347
4.10
Chlorpyrifos
5.602
NS*
4.00
Phosfolan
5.420
NS*
4.00
Stirophos
4.602
NS 1
4.00
not significant. compared for their inhibitory effect to acetyl cholinesterase (AChE), neurotoxic esterase (NIE), and monoamine oxidase (MAO) as shown in table 1. The five OP compounds exerted different degrees of inhibition to AChE, and the most potent inhibitor was chlorpyriitos , followed by EPN, phosfolan, Leptophos, and stirophos in a descending order* On the other hand, Leptophos was almost 100 fold as potent to both NTE and MAO followed by EPN while the other three candidate compounds did not show any apprecibable inhibition of either NTE or MAO.
This clear specific affinity of
NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES
281
Leptophos or EPN for blocking NTE or MAO must be of significance and should be taken i n consideration in any interpretation of the characteristic effect of these two compounds in inducing symptoms of delayed neurotoxicity. In recent reports, in-vitro inhibition of MTE i s taken as a precise criterion in detecting the delayed neurotoxic
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agents ' .
Thus evidence began to support the impression
that acetylcholinesterase inhibition i s not the solely mode of toxic action for OP compounds especially those capable of causing delayed neurotoxicity. If the chemical structure i s considered, i t can also be deduced that the phosphonate structure in Leptophos and EPN has a positive correlation with either delayed neurotoxicity or NTE and MAO inhibition.
Chlorpyrifos , phosfolan
and stirophos did not show neither delayed neurotoxicity symptoms nor specific inhibition of NTE or MAO. The Mode of Delayed Neurotoxic Action for Some Órgano— phosphorus Compounds : The common belief was that organophosphorus compounds generally exert their acute toxicity through their ability to inhibit the cholinesterase i n the nervous system. Yet some of these OP esters are characterized by being capable of inducing irreversible chronic delayed neurotoxicity as was shown by TOCP, Mepafox, leptophos and EPN. The machanism of this chronic delayed neurotoxicity
cannot
be interpretted according to their effect as ChE inhibitors. I t was interesting that in the rat liver, isocarbonazid amidase was shown to be a more sensitive indicator of
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exposure to EPN intoxication than serum cholinesterase 18 inhibition . This assures the fact that there are other biological sites which are highly affected by OP compounds. Early detection of delayed neurotoxic agents depends upon histopathological examination showing predominantly degeneration of the fatty myelin sheath surrounding long Downloaded by [ECU Libraries] at 01:15 21 April 2015
nerve axons.
Therefore, the term demylinating agents
was used to differentiate such compounds. However, later work showed that the nerve axon itself was primarily affected and damage to myelin was only a secondary effect °. Recently, i t was observed that the chronic ataxia was accomanied by the inhibition of the brain neurotoxic esterase ' .
However, the actual role of the brain
neurotoxic esterase inhibition in exerting delayed neurotoxicity i s s t i l l vague, and the chain of events from this initial alteration of the protein to degeneration of the nerve axon i s s t i l l unknown. Besides, the function of this phosphorylated protein responsible for neurotoxic esterase activity in the brain i s s t i l l also obscure. This situation necessitates and suggests the following postulation.
The chronic ataxia and irreversible para-
lysis of the hind legs i s expected to take place due to lesion in the central nervous system which i s directly connected to the spinal cord and the sciatic nerve. Evidence i s accumulating to prove that both adrenergic and cholinergic systems are interacting at the synapases of the neuromuscular junctions.
NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES
283
It was reported that the sympathetic neurons at the brain adrenal medulla liberate acetylcholine which in turn evokes a secretion of adrenaline and noradrenaline
20
.
Recently, i t was shown that two types of cholinergic receptors-muscarinic and nicotinic- are present on adrenergic nerve terminals in sympathetic effector organs.
Clear
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direct evidence for prejunctional nicotinic receptors came from the findings that in prefused organs administration of nicotine or acetylcholine (ACh) resulted in the release of norepinephrine (NE) and in the initiation of antidromic action potentials in sympathetic nerve fibers to the organs.
Similar evidence for prejunctional muscarinic
receptors on adrenergic nerve terminals was also shown. The activation of these receptors by ACh and methacholine at low concentrations inhibited the release of (NE) evoked either by nerve stimulation or by stimulation of the neuronal nicotinic receptors, i t was also concluded that in the central nervous system, presynaptic inhibitory muscarinic receptors may well be present on dopaminergic as well as adrengergic terminals 21 . In another study i t was concluded that activation or impairment of dapaminergic transmission results in opposite effects on the release of ACh from the striatum. That mutual influence of DA and ACh neurons in the extrapyramidal centers was considered of both physiological and patho— physiological reflections22.
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AL.
Parallel to this correlation, a recent study had shown the implication of the dopaminergic activity on the dorsal and ventral locomotor activity in the r a t . I t was found that selective and extensive destruction of the nigroneostriatal DA projection, so that neostriatal DA levels were reduced to 7.5% of controls, resulted in a profound
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attenuation but not a complete blockage of the locomotor stimulant effect of D-emphetamine. Besides, the possibility that norepinepherine in the cerebellum or midbrain may mediate some of the stimulant effects of amphetamine cannot be excluded •*. All these interactions support the conclusion that the delayed neurotoxic agents should not be considered solely as anticholinesterases.
They are expected to have
a multitoxic mechanism in which their inhibitory effect will interact simultaneously with adrenergic, dopaminergic and cholinergic sites.
The released ACb during the acute
toxicity of these OP toxicants might induce and activate adrenergic and dopaminergic agents.
The concentration of
liberated dopamine, epinepherine, and other biogenic amines will also be continuously increased by the particular ability of delayed neurotoxic agents to act as MAO
inhibitors
resulting in free release of biogenic amines at the adrenergic terminals which are highly abundant in the CHS the spinal cord in mammals.
and
These free adrenogenic
transmitters are strong intact poisons.
When they diffuse
to bind to their specific receptors on the postsynaptic membrane they will increase permeability to ions, with
NEUROTOXICITY OF ORGANOPHOSPHORUS INSECTICIDES
285
increased metabolic processes but reduced protein biosynthesis.
This reduction in protein biosynthesis and the
depletion of ions might represent an important cause of the lesions caused in the neuroaxons
'
.
The increased
neuromembrane permeability might lead to liberation of cellular hydrolases or complex changes of molecular
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architecture of membrane-bound enzymes and the interference with oxidative processes at the level of the neuronal cytoplasm and organelles as well as with energy transference and utilization mechanism. All these effects might be included as possible causes of irreversible neurotoxic effects.
This interpretation i s generally
supported by the present data showing that leptophos and EPN are characterized by being strong monoamine oxidase inhibitors as well as potent delayed neurotoxic agents. Their effect as neurotoxic esterase inhibitors might represent one of the steps in blocking the protein synthesis reactions. This postulated mechanism draws the attention to the importance of considering the adrenergic and dopaminergic neurone as the main c r i t i c a l sites of toxic action for the delayed neurotoxic agents in mamnals. The long required stepwise mechanism of such chain interactions might explain the elapsed time required to reach the delayed symptoms of those neurotoxic agents. Also the same complex mechanism leads to the irreversible lesions in the central nervous system.
The chemical
stability, persistance and high lipophiliclty of the
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two compounds Leptophos and EPîï support t h e i r ability to exist i n active form long enough to exert the delayed neurotoxic effects in the CNS of mammals and birds.
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ACKNOWLEDGEMENT
This work was p a r t i a l l y supported by a grant from the project (PR 3-545-1) financed by t h e U.S.E.P.A. Their assistance and cooperation i s highly appreciated.
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