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Neurotoxicologic investigations of the pesticide dichlorvos (DDVP). Effects on the central and peripheral nervous system
Toxicology, 49 (1988) 141-148
Elsevier ScientificPublishers Ireland Ltd.
N E U R O T O X I C O L O G I C I N V E S T I G A T I O N S OF T H E P E S T I C I D E DICHLORVOS (DDVP). E F F E C T S ON T H E C E N T R A L AND P E R I P H E R A L NERVOUS SYSTEM*
ILLI~SDESI and LASZL0 NAGYMAJT]~NYI Department of Hygiene and Epidemiology, Albert Szent-Gy6rgyi University Medical Schoo4 H-67~0 D&n tdr 10 Szeged (Hungary)
SUMMARY The neurotoxic effect on the central and peripheral nervous system of dichlorvos (DDVP) was investigated by a computer system in acute and subchronic experiments in CFY male rats. The administered peroral doses were given by gavage; the acute group was given a single 88 mg/kg dose and the 2 subchronic groups were given 1.6 mg/kg or 0.8 mg/kg daily for a period of 6 weeks. Significant changes of the function of CNS - increase of EEG mean frequency, decrease of EEG mean amplitude, that of activity of EEG bands (power density) -- and peripheral nervous system -- decrease of conduction velocity, increase of relative and absolute refractory periods -- were found after t r e a t m e n t with both the single large and repeated small doses of dichlorvos. There were no correlations between the functional disturbances of the central and peripheral nervous systems and the inhibition of the cholinesterase activity in various organs and the blood.
words: Dichlorvos (DDVP); Electroencephalography; Electroneuromyography; CNS; Peripheral nervous system; Cholinesterase-activity; Neurotoxicity
Key
INTRODUCTION Dichlorvos (DDVP) is an organic phosphate ester that is used world-wide in agriculture, public health and consumer households as a contact and stomach insecticide. It is moderately to highly toxic for animals, highly toxic for man because of its direct inhibitory effect on the cholinesterase activity *According to the lecture held at the first International NeurotoxicologyAssociation Meeting Lunteren, The Netherlands, 1987. 0300483X/88/$03.50 © 1988 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
141
in the nervous system and other tissues. The signs of intoxication, i.e. salivation, diarrhoea, tremors, convulsions, appear in parallel with the decrease of the enzyme activity. Dichlorvos does not produce delayed neurotoxicity in hens, but some neurotoxic effects, mild signs of ataxia with or without inhibition of brain and spinal cord esterase, were found after giving a single large dose [1--5]. According to Hyde et al. [6], a lethal dose of DDVP did not produce electrocortical disturbances in male rats, and no changes of nerve conduction velocity was found by Hazelwood et al. [7] in dogs treated with a high dose of the chemical although serious signs of intoxication were seen in both the above experiments. To obtain detailed information and to solve the nuclear problems about the neurotoxicologic effect of dichlorvos intoxication we used a sophisticated computer controlled system for the study of the early, subtle and subclinical influences on the central nervous system as well as on the peripheral neuromuscular system in both acute and subchronic experiments. METHODS
Animals Male CFY rats with a body mass between 200 and 250 g, 8 rats in each group. Housing Under standard laboratory conditions. Material Dichlorvos: 2,2-dichlorovinyl-dimethylphosphate, active material of 98.6% purity. Fabricated by Universal Laboratories of Szeged, Hungary. Treatment Acute experiment - 88 mg/kg of dichlorvos peroral through a gavage on one occasion, examinations 24 h later. Subchronic experiments - EEG: 1.6 mg/kg of DDVP peroral through a gavage daily for 6 weeks. Peripheral neuromuscular system: 1.6 mg/kg and 0.8 mg/kg of dichlorvos peroral, respectively, every day for 6 weeks. EEG examinations [8] Silver electrodes were placed frontally and parieto-occipitally on the skull of male CFY rats. To examine the alert freely moving rats a 12-channel EEG apparatus (type EEG 14, made by EMG of Esztergom, Hungary) was used. The EEG activity was resolved into 6 different frequency bands by means of an on-line method applying Fourier analysis through band pass filters. The EEG apparatus was connected to a ZX Spectrum analyser and a Commodore 128 computer system. For each of the analysed periods (20 s) the following parameters were calculated: (a) EEG mean frequency; (b) mean amplitude
142
values of the complex EEG recordings as well as the extent of the participation of each band in the composition of the complex curve (power density); and (c) an index number formed by the proportion of the slow (d + e} vs. the fast (/31 + f~2)wave bands. The slowing of bioelectrical activity of the brain, representative of a decrease in central excitation level is indicated by the increase of this index number, while its diminution is caused by the preponderance of the fast components and thus shows the increase in the excitation level of the central nervous system. With the help of (b) and (e) the EEG response could be evaluated much more precisely than by determining (a) alone. In addition (c) reduces EEG activity to one figure so it can be easily analysed.
Examination of conduction velocity of the peripheral nerve [9] This examination was carried out by stimulating the nerve in the rat's tail. Supramaximal stimuli were used, and they were chosen so that their increase did not increase further the amplitude of the net response impulse. Since conduction velocity of the nerve is dependent on temperature, the tail was continuously maintained at 25 °C.
Determination of refractory periods [10] Electrodes were placed as mentioned above. The nerve was stimulated by double impulses with gradual decreases in the interval between the 2 impulses (At = 10 ms, 3 ms, etc.), until the response latency to the second stimulus increased in parallel to the decrease in the inter stimulus interval (change in the relative refractory period), or until the response to the second stimulus disappeared (absolute refractory period), respectively. The relative and absolute refractory periods were determined by the computer system using the L 1 and L 2 latency times (Lx: latency time after the first stimulus, L2: latency time after the second stimulus) and the t intervals.
Computer software Our computer system is built on a microprocessor system that has 13 inputs (EEG, EMG, ECG, etc.). Any one of these inputs can take up to 1.024 incoming samples, and the time and the parameters of amplification can be defined by a program that has been specially written for this purpose. The data are put into the memory of the computer, then a signal analysis on the first level mathematical-statistical computation is done. Its results and/or the samples can be visualised on a screen or written out on a printer. A Commodore 128, which is connected to the system, does the computation of the mean values and on the basis of them it determines the statistical calculation.
Examinations of the cholinesterase activity This was carried out according to the method of Meinecke and Oettel [11] from red blood cells, plasma, brain cortex, white matter, liver, heart muscle, and skeletal muscle. In the acute experiments the values of the experimental
143
rats were compared to that of the untreated control. In the subchronic experiments different groups of DDVP treated rats, with 8--8 in the experiment and 8 - 8 in the control, were sacrificed at the end of the first, second, fourth and sixth week. At the end of the sixth week the remainder on which the neurotoxicological investigations had been performed were killed and the results were compared with the data of untreated rats.
Mathematical computations The experimental results were analysed on a Commodore 128 computer. Evaluation where necessary was made using Student's 2-sample t-test. RESULTS We found in our preliminary experiments a peroral LD~ for male rats was 88 mg/kg. In response to the high dose in the acute experiments general symptoms of organic phosphate intoxication occurred including salivation, muscle fibrillation and cramps. Since some animals died after t r e a t m e n t with
1 a/EEO
MEAN FREQUENCY
Hz { - - t BEFORE TREATMENT
lc/EEG INDEX ~+~
8
2/, HOURS AFTER DDVP 88 rng/kg p.o.
7
~/
~
/~¢*/~;'
1.3 1.2 11 10 09 08 07 0.6 .}. 05,
6
,•PLACING OF ELECTRODES ON THE RAT'S CALVARIA
mvZ 110' 100 90' 80"
1 b/MEAN
+
-t-
70" 60'
50' 40' 3O 2O 10 COMPLEX EEG
1.5-3
5
AMPLITUDES OF EEG- BANDS
I 17 o 4-7
o~ 8-13
n
14-20
I i
~ ~
I~
H3C-O O H3C- 0
H cl CI
DICHLORVOS(DDVP) p.o LO50 88rng/kg
21-30
IF EEG-BANDS 31-70 Hz
Fig. 1. The effect of DDVP treatment on the EEG of awake, freely moving male CFY rats in acute experiments. 1 (a), 1 (c) blank circles: before treatment; lined circles: after treatment. 1 (b)
ordinate: in per cent of control mV values.
144
high doses, (the teatment started with 20 animals/group) in the end 8 animals/group were studied and evaluated. No clinical symptoms were detected in the subchronic condition. The effects on the central nervous system and peripheral nervous system of acute administration of a high dose of DDVP, or those of subchronic treatment of small doses, respectively, are presented in Figs. I through 5. As shown in the acute EEG diagram (Fig. 1} the mean frequency increased (Fig. la), while the participation ratio of single EEG bands diminished (Fig. lb). The EEG index decreased (Fig. lc), which is regarded as a sign of greater central excitation. In the subchronic experiments there were considerable increases in the EEG frequency at the end of the first week, and there was a slight decrease afterwards, but the mean frequency still remained faster than the initial value, even after 6 weeks (Fig. 2a) the participation ratios of single EEG bands diminished here, too (Fig. 2b). There was also a strong diminution in the EEG index on the first week - which means an increase of the central excitation level -- followed by a slight
2 a/EEG
NEAN FREQUENCY
~4.._~.__ StOW ce~r~,.a~fs /5(*/3 z - fost - ' " " .... increosing ",, diminishing "
.,dammoshes
CNS excitobility ~-
0ncreases
2 c/EEG INDEX
mVX
"2 b/MEAN AMPUTUDES OF EEG- BANDS
110 100 90
1.1 1.0 0.9 0.8 0.7 0.6 0.5 0£
80 70 60
40 30 20 10
Weeks
01246 01246 $ COMPLEX EEG 15-3
01246
01 246
1246
4-7
8 - 13
14- 20
/~0
0124 ~ 21-30
01246 WEEKS T EEG-BANDS 31-70 Hz
Fig. 2. The effect of DDVP treatment on the EEG of awake, freely moving rats in subchronic experiments. 2 (a) E E G mean frequency; 2 (b) mean amplitudes of the EEG complex and of different frequency bands (power density); 2 (c) E E G index. In frame: E E G index, diminishing value indicates the increase of the central nervous excitability and increasing value means the decrease of CNS excitability.
145
Vll,4 DDVP 1.6 mg/kg p o daily I
I Control
* p < 0.01
DDVP 0.8 mg/kg po daily
~
m/sec 2/. 22 20 18 16. 14'
+
+ I/z x I I (X
t<
12' 108, 6 4 2
i
÷ //
p < 0001
d~stence of conducting conduchon velocffy(m/sec) electrodes m mm dJfference between latency hines in msec after stimuli
..
I/
/I
I I ('x
/ 1 X)I
, ,
tlX)l
(X
I1( x ii(x II ,v //X /iX)
~1 7~1 / i X)! t i ×)1 i i Xlt //x,1
O(
2
4
O(
shmulahng electrodes
conducting
kq
electrodes
i
tQil
nerve
J
latency times msec
6
Weeks
Fig. 3. The effect of DDVP t r e a t m e n t of rats on the conduction velocity of the tail nerve in subchronic experiments.
increase on the second to fourth weeks and again a decrease of the curve -i.e. signs of decrease and increase respectively of central excitation levels -on the last week (Fig. 2c). No changes were shown in the conduction velocity of the peripheral nerve in acute experiments. While significant decreases were found in the subchronic ones (Fig. 3). The decrease in conduction velocity was, except for the sixth week, dose dependent. Remarkable rises were evident in the relative and absolute refractory periods of the peripheral nerve in both the acute and subchronic examinations (Figs. 4 and 5). msec
2.4 22 20 18 16 1/. 12 10 08 06 04 0.2
I
EZ3 Control
II
2z. hours after
T
DDVP 88 mg/kg po
msec /~/,~/ /// ii/
5O ~5
~o
III
35
///
30
III
25
I/I
._.
/~/ I/I
**
p < 0001
~//
10 I
O5
Fig. 4. The effect of DDVP t r e a t m e n t of rats on the absolute (I) and relative (II) refractory period of the tail nerve in acute experiments.
146
EZZZ]DDVP 1.6mg/kg po do,ty
I---'1 Controt
p<0001
DDVP 08mg/kg podody
rnsec
II.
I
2,8 2.6
2/..
:
2.2 2,0 18
÷
1.6 1.4 1.2
/ /
/ / V
v
1.o
/
0.8. 0.50.4. 0.2.
/ // / ,//
1
2
/.
6
2
4
6 ~e-~s
Fig. 5. The effect of DDVP t r e a t m e n t of rats on the absolute (I) and relative (II) refractory period of the tail nerve in subchronic experiments.
In the acute experiments both the relative and the absolute refractory periods became significantly longer. The same was true in the case of the subchronic experiments where both types of refractory periods became longer and dose dependent. The inhibition of cholinesterase activity of red blood cells, plasma and different tissues (cerebral cortex, white matter of the brain, liver and heart muscle) except that of the skeletal muscles, was significantly stronger in acute experiments than that of the control, untreated rats. The trend of the changes of cholinesterase activity in examined organs and the blood were different during the subchronic experiments in the different groups of DDVP-treated animals at the end of the first, second, fourth and sixth weeks, respectively, when compared to the values of the control rats. DDVP administration produced a significantly decreased, but finally somewhat improving enzyme activity in the cerebral cortex, liver and heart muscle. We found a continuous depression in white matter and stepwise diminution of activity in plasma. The activity in red blood cells and skeletal muscles fluctuated during the whole period of treatment. DISCUSSION
The high dose in the acute experiments produced all the well-known symptoms of organophosphate intoxication. The changes in investigated parameters of central and peripheral nervous systems were remarkable. The increase of the EEG mean frequency, the activity changes of the EEG bands, the decrease of the EEG index suggested an enhanced excitation of the
147
c e n t r a l n e r v o u s s y s t e m . This d i s o r d e r in the c e r e b r a l function was p r o b a b l y due to the large a m o u n t s of D D V P and could be a t t r i b u t e d to the d e c r e a s i n g of the cholinesterase a c t i v i t y of the c e r e b r a l c o r t e x and white m a t t e r . As a c o n s e q u e n c e of a l t e r e d e n z y m e activity, acetylcholine c o n c e n t r a t i o n s p r o b a b l y i n c r e a s e d in all brain r e g i o n s [12] causing d i s t u r b a n c e s in the function of t h e c e n t r a l n e r v o u s s y s t e m . A s the conduction velocity did not show any c h a n g e s a single, although high dose of DDVP, s e e m e d to be insufficient to s e v e r e l y d a m a g e the conduction in p e r i p h e r a l n e r v e s . The increase of the absolute and relative r e f r a c t o r y periods, h o w e v e r , points to a subtle a l t e r a t i o n of its function. The d a t a of t h e subchronic e x p e r i m e n t s p r o v e d t h a t r e l a t i v e l y small doses of D D V P e x e r t e d a d e t e r i o r a t i n g effect on t h e functions of the central and p e r i p h e r a l n e r v o u s s y s t e m e v e n a f t e r a s h o r t period. I t is v e r y i m p o r t a n t , t h a t w i t h o u t a n y clinical s y m p t o m s of intoxication t h e functional p a r a m e t e r s of the central and p e r i p h e r a l n e r v o u s s y s t e m s , i.e. E E G m e a n frequency, E E G index, conduction velocity, r e f r a c t o r y periods w e r e altered. It is n o t e w o r t h y too, t h a t t h e r e w e r e no direct correlations b e t w e e n the obtained a l t e r a t i o n s of t h e c e n t r a l and p e r i p h e r a l n e r v o u s s y s t e m on t h e one hand and c h a n g e s of t h e c h o l i n e s t e r a s e activity of blood and o t h e r tissues on the o t h e r hand. W e believe t h a t our m e t h o d s d e m o n s t r a t e d here would be suitable to indicate e a r l y and mild c h a n g e s of t h e sensitive functions of the o r g a n i s m and t h u s will be applicable for t h e p r e v e n t i o n of p e r m a n e n t d a m a g e . REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12
148
W.A. Aldridge and M.K. Johnson, Side effects of organophosphorous compounds: delayed neurotoxicity. WHO Bull., 44 (1971) 259. M.K. Johnson, The anomalous behavior of dimethyl phosphates in the biochemical test for delayed neurotoxicity. Arch. Toxicol., 41 (1978) 107. M.K. Johnson, Delayed neurotoxicity: do trichlorphon and/or dichlorvos cause delayed neurpathy in man or in test animals? Acta Pharmacol. Toxicol., Suppl. 5, 49 (1971) 87. S. Caroldi and M. Lotti, Delayed neurotoxicity caused by a single massive dose of diehlorvos to adult hens. Toxieol. Lett., 9 (1981) 157. B.M. Francis, P.L. Metcalf and L.G. Hansen, Toxicity of organophosphorus esters to laying hens after oral and dermal administration. J. Environ. Sci. Health, B20(1) (1985) 73. K.M. Hyce, J.C. CrandaU, K.E. Kortman, and W.K. McCoy, EEG, ECG and respiratory response to acute insecticide exposure. Bull. Environ. Contain. Toxicol., 19 (1978) 47. J.C. Hazelwood, G.E. Stefan and J.M. Bowen, Motor unit irritability in beagles before and after exposure to cholinesterase inhibitors. Am. J. Vet. Res., 41(6) (1979) 852. I. D6si, Judit Szlobodnyik, M~ria Karmos and ~gnes Strohmayer, Neurotoxicological investigation of a new Hungarian pesticide Toxurazine. Acta Physiol. Acad. Sci. Hung., 60 (1982) 1. T. Miyoshi and I. Goto, Serial in vivo determinations of nerve conduction velocity in rat tails. Electroeneephalogr. Clin. Neurophysiol., 35 (1973) 125. Elisabeth Anda, Gy. Dura, I. L6rincz, I. Vincze and Magdolna Kert~sz, The effect of CO2 on the central nervous system (in Hungarian), Eg6szs6gtudom~ny, 28 (1984) 270. K.H. Meinecke and H. Oettel, Mikromethode zur Bestimmung der Acethylcholinesterase Aktivit~t in Erythrocyten und Plasma von Menseh und Tier. Arch. Toxicol., 22 (19671244. A.T. Modak, W.B. Stavihona and S.T. Weintraub, Dichlorvos and the cholinergic system: effects on cholinesterase and acetylcholine and choline contents of rat tissues. Arch. Int. Pharmacodyn. Ther., 217 (1975) 293.
Elsevier ScientificPublishers Ireland Ltd.
N E U R O T O X I C O L O G I C I N V E S T I G A T I O N S OF T H E P E S T I C I D E DICHLORVOS (DDVP). E F F E C T S ON T H E C E N T R A L AND P E R I P H E R A L NERVOUS SYSTEM*
ILLI~SDESI and LASZL0 NAGYMAJT]~NYI Department of Hygiene and Epidemiology, Albert Szent-Gy6rgyi University Medical Schoo4 H-67~0 D&n tdr 10 Szeged (Hungary)
SUMMARY The neurotoxic effect on the central and peripheral nervous system of dichlorvos (DDVP) was investigated by a computer system in acute and subchronic experiments in CFY male rats. The administered peroral doses were given by gavage; the acute group was given a single 88 mg/kg dose and the 2 subchronic groups were given 1.6 mg/kg or 0.8 mg/kg daily for a period of 6 weeks. Significant changes of the function of CNS - increase of EEG mean frequency, decrease of EEG mean amplitude, that of activity of EEG bands (power density) -- and peripheral nervous system -- decrease of conduction velocity, increase of relative and absolute refractory periods -- were found after t r e a t m e n t with both the single large and repeated small doses of dichlorvos. There were no correlations between the functional disturbances of the central and peripheral nervous systems and the inhibition of the cholinesterase activity in various organs and the blood.
words: Dichlorvos (DDVP); Electroencephalography; Electroneuromyography; CNS; Peripheral nervous system; Cholinesterase-activity; Neurotoxicity
Key
INTRODUCTION Dichlorvos (DDVP) is an organic phosphate ester that is used world-wide in agriculture, public health and consumer households as a contact and stomach insecticide. It is moderately to highly toxic for animals, highly toxic for man because of its direct inhibitory effect on the cholinesterase activity *According to the lecture held at the first International NeurotoxicologyAssociation Meeting Lunteren, The Netherlands, 1987. 0300483X/88/$03.50 © 1988 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
141
in the nervous system and other tissues. The signs of intoxication, i.e. salivation, diarrhoea, tremors, convulsions, appear in parallel with the decrease of the enzyme activity. Dichlorvos does not produce delayed neurotoxicity in hens, but some neurotoxic effects, mild signs of ataxia with or without inhibition of brain and spinal cord esterase, were found after giving a single large dose [1--5]. According to Hyde et al. [6], a lethal dose of DDVP did not produce electrocortical disturbances in male rats, and no changes of nerve conduction velocity was found by Hazelwood et al. [7] in dogs treated with a high dose of the chemical although serious signs of intoxication were seen in both the above experiments. To obtain detailed information and to solve the nuclear problems about the neurotoxicologic effect of dichlorvos intoxication we used a sophisticated computer controlled system for the study of the early, subtle and subclinical influences on the central nervous system as well as on the peripheral neuromuscular system in both acute and subchronic experiments. METHODS
Animals Male CFY rats with a body mass between 200 and 250 g, 8 rats in each group. Housing Under standard laboratory conditions. Material Dichlorvos: 2,2-dichlorovinyl-dimethylphosphate, active material of 98.6% purity. Fabricated by Universal Laboratories of Szeged, Hungary. Treatment Acute experiment - 88 mg/kg of dichlorvos peroral through a gavage on one occasion, examinations 24 h later. Subchronic experiments - EEG: 1.6 mg/kg of DDVP peroral through a gavage daily for 6 weeks. Peripheral neuromuscular system: 1.6 mg/kg and 0.8 mg/kg of dichlorvos peroral, respectively, every day for 6 weeks. EEG examinations [8] Silver electrodes were placed frontally and parieto-occipitally on the skull of male CFY rats. To examine the alert freely moving rats a 12-channel EEG apparatus (type EEG 14, made by EMG of Esztergom, Hungary) was used. The EEG activity was resolved into 6 different frequency bands by means of an on-line method applying Fourier analysis through band pass filters. The EEG apparatus was connected to a ZX Spectrum analyser and a Commodore 128 computer system. For each of the analysed periods (20 s) the following parameters were calculated: (a) EEG mean frequency; (b) mean amplitude
142
values of the complex EEG recordings as well as the extent of the participation of each band in the composition of the complex curve (power density); and (c) an index number formed by the proportion of the slow (d + e} vs. the fast (/31 + f~2)wave bands. The slowing of bioelectrical activity of the brain, representative of a decrease in central excitation level is indicated by the increase of this index number, while its diminution is caused by the preponderance of the fast components and thus shows the increase in the excitation level of the central nervous system. With the help of (b) and (e) the EEG response could be evaluated much more precisely than by determining (a) alone. In addition (c) reduces EEG activity to one figure so it can be easily analysed.
Examination of conduction velocity of the peripheral nerve [9] This examination was carried out by stimulating the nerve in the rat's tail. Supramaximal stimuli were used, and they were chosen so that their increase did not increase further the amplitude of the net response impulse. Since conduction velocity of the nerve is dependent on temperature, the tail was continuously maintained at 25 °C.
Determination of refractory periods [10] Electrodes were placed as mentioned above. The nerve was stimulated by double impulses with gradual decreases in the interval between the 2 impulses (At = 10 ms, 3 ms, etc.), until the response latency to the second stimulus increased in parallel to the decrease in the inter stimulus interval (change in the relative refractory period), or until the response to the second stimulus disappeared (absolute refractory period), respectively. The relative and absolute refractory periods were determined by the computer system using the L 1 and L 2 latency times (Lx: latency time after the first stimulus, L2: latency time after the second stimulus) and the t intervals.
Computer software Our computer system is built on a microprocessor system that has 13 inputs (EEG, EMG, ECG, etc.). Any one of these inputs can take up to 1.024 incoming samples, and the time and the parameters of amplification can be defined by a program that has been specially written for this purpose. The data are put into the memory of the computer, then a signal analysis on the first level mathematical-statistical computation is done. Its results and/or the samples can be visualised on a screen or written out on a printer. A Commodore 128, which is connected to the system, does the computation of the mean values and on the basis of them it determines the statistical calculation.
Examinations of the cholinesterase activity This was carried out according to the method of Meinecke and Oettel [11] from red blood cells, plasma, brain cortex, white matter, liver, heart muscle, and skeletal muscle. In the acute experiments the values of the experimental
143
rats were compared to that of the untreated control. In the subchronic experiments different groups of DDVP treated rats, with 8--8 in the experiment and 8 - 8 in the control, were sacrificed at the end of the first, second, fourth and sixth week. At the end of the sixth week the remainder on which the neurotoxicological investigations had been performed were killed and the results were compared with the data of untreated rats.
Mathematical computations The experimental results were analysed on a Commodore 128 computer. Evaluation where necessary was made using Student's 2-sample t-test. RESULTS We found in our preliminary experiments a peroral LD~ for male rats was 88 mg/kg. In response to the high dose in the acute experiments general symptoms of organic phosphate intoxication occurred including salivation, muscle fibrillation and cramps. Since some animals died after t r e a t m e n t with
1 a/EEO
MEAN FREQUENCY
Hz { - - t BEFORE TREATMENT
lc/EEG INDEX ~+~
8
2/, HOURS AFTER DDVP 88 rng/kg p.o.
7
~/
~
/~¢*/~;'
1.3 1.2 11 10 09 08 07 0.6 .}. 05,
6
,•PLACING OF ELECTRODES ON THE RAT'S CALVARIA
mvZ 110' 100 90' 80"
1 b/MEAN
+
-t-
70" 60'
50' 40' 3O 2O 10 COMPLEX EEG
1.5-3
5
AMPLITUDES OF EEG- BANDS
I 17 o 4-7
o~ 8-13
n
14-20
I i
~ ~
I~
H3C-O O H3C- 0
H cl CI
DICHLORVOS(DDVP) p.o LO50 88rng/kg
21-30
IF EEG-BANDS 31-70 Hz
Fig. 1. The effect of DDVP treatment on the EEG of awake, freely moving male CFY rats in acute experiments. 1 (a), 1 (c) blank circles: before treatment; lined circles: after treatment. 1 (b)
ordinate: in per cent of control mV values.
144
high doses, (the teatment started with 20 animals/group) in the end 8 animals/group were studied and evaluated. No clinical symptoms were detected in the subchronic condition. The effects on the central nervous system and peripheral nervous system of acute administration of a high dose of DDVP, or those of subchronic treatment of small doses, respectively, are presented in Figs. I through 5. As shown in the acute EEG diagram (Fig. 1} the mean frequency increased (Fig. la), while the participation ratio of single EEG bands diminished (Fig. lb). The EEG index decreased (Fig. lc), which is regarded as a sign of greater central excitation. In the subchronic experiments there were considerable increases in the EEG frequency at the end of the first week, and there was a slight decrease afterwards, but the mean frequency still remained faster than the initial value, even after 6 weeks (Fig. 2a) the participation ratios of single EEG bands diminished here, too (Fig. 2b). There was also a strong diminution in the EEG index on the first week - which means an increase of the central excitation level -- followed by a slight
2 a/EEG
NEAN FREQUENCY
~4.._~.__ StOW ce~r~,.a~fs /5(*/3 z - fost - ' " " .... increosing ",, diminishing "
.,dammoshes
CNS excitobility ~-
0ncreases
2 c/EEG INDEX
mVX
"2 b/MEAN AMPUTUDES OF EEG- BANDS
110 100 90
1.1 1.0 0.9 0.8 0.7 0.6 0.5 0£
80 70 60
40 30 20 10
Weeks
01246 01246 $ COMPLEX EEG 15-3
01246
01 246
1246
4-7
8 - 13
14- 20
/~0
0124 ~ 21-30
01246 WEEKS T EEG-BANDS 31-70 Hz
Fig. 2. The effect of DDVP treatment on the EEG of awake, freely moving rats in subchronic experiments. 2 (a) E E G mean frequency; 2 (b) mean amplitudes of the EEG complex and of different frequency bands (power density); 2 (c) E E G index. In frame: E E G index, diminishing value indicates the increase of the central nervous excitability and increasing value means the decrease of CNS excitability.
145
Vll,4 DDVP 1.6 mg/kg p o daily I
I Control
* p < 0.01
DDVP 0.8 mg/kg po daily
~
m/sec 2/. 22 20 18 16. 14'
+
+ I/z x I I (X
t<
12' 108, 6 4 2
i
÷ //
p < 0001
d~stence of conducting conduchon velocffy(m/sec) electrodes m mm dJfference between latency hines in msec after stimuli
..
I/
/I
I I ('x
/ 1 X)I
, ,
tlX)l
(X
I1( x ii(x II ,v //X /iX)
~1 7~1 / i X)! t i ×)1 i i Xlt //x,1
O(
2
4
O(
shmulahng electrodes
conducting
kq
electrodes
i
tQil
nerve
J
latency times msec
6
Weeks
Fig. 3. The effect of DDVP t r e a t m e n t of rats on the conduction velocity of the tail nerve in subchronic experiments.
increase on the second to fourth weeks and again a decrease of the curve -i.e. signs of decrease and increase respectively of central excitation levels -on the last week (Fig. 2c). No changes were shown in the conduction velocity of the peripheral nerve in acute experiments. While significant decreases were found in the subchronic ones (Fig. 3). The decrease in conduction velocity was, except for the sixth week, dose dependent. Remarkable rises were evident in the relative and absolute refractory periods of the peripheral nerve in both the acute and subchronic examinations (Figs. 4 and 5). msec
2.4 22 20 18 16 1/. 12 10 08 06 04 0.2
I
EZ3 Control
II
2z. hours after
T
DDVP 88 mg/kg po
msec /~/,~/ /// ii/
5O ~5
~o
III
35
///
30
III
25
I/I
._.
/~/ I/I
**
p < 0001
~//
10 I
O5
Fig. 4. The effect of DDVP t r e a t m e n t of rats on the absolute (I) and relative (II) refractory period of the tail nerve in acute experiments.
146
EZZZ]DDVP 1.6mg/kg po do,ty
I---'1 Controt
p<0001
DDVP 08mg/kg podody
rnsec
II.
I
2,8 2.6
2/..
:
2.2 2,0 18
÷
1.6 1.4 1.2
/ /
/ / V
v
1.o
/
0.8. 0.50.4. 0.2.
/ // / ,//
1
2
/.
6
2
4
6 ~e-~s
Fig. 5. The effect of DDVP t r e a t m e n t of rats on the absolute (I) and relative (II) refractory period of the tail nerve in subchronic experiments.
In the acute experiments both the relative and the absolute refractory periods became significantly longer. The same was true in the case of the subchronic experiments where both types of refractory periods became longer and dose dependent. The inhibition of cholinesterase activity of red blood cells, plasma and different tissues (cerebral cortex, white matter of the brain, liver and heart muscle) except that of the skeletal muscles, was significantly stronger in acute experiments than that of the control, untreated rats. The trend of the changes of cholinesterase activity in examined organs and the blood were different during the subchronic experiments in the different groups of DDVP-treated animals at the end of the first, second, fourth and sixth weeks, respectively, when compared to the values of the control rats. DDVP administration produced a significantly decreased, but finally somewhat improving enzyme activity in the cerebral cortex, liver and heart muscle. We found a continuous depression in white matter and stepwise diminution of activity in plasma. The activity in red blood cells and skeletal muscles fluctuated during the whole period of treatment. DISCUSSION
The high dose in the acute experiments produced all the well-known symptoms of organophosphate intoxication. The changes in investigated parameters of central and peripheral nervous systems were remarkable. The increase of the EEG mean frequency, the activity changes of the EEG bands, the decrease of the EEG index suggested an enhanced excitation of the
147
c e n t r a l n e r v o u s s y s t e m . This d i s o r d e r in the c e r e b r a l function was p r o b a b l y due to the large a m o u n t s of D D V P and could be a t t r i b u t e d to the d e c r e a s i n g of the cholinesterase a c t i v i t y of the c e r e b r a l c o r t e x and white m a t t e r . As a c o n s e q u e n c e of a l t e r e d e n z y m e activity, acetylcholine c o n c e n t r a t i o n s p r o b a b l y i n c r e a s e d in all brain r e g i o n s [12] causing d i s t u r b a n c e s in the function of t h e c e n t r a l n e r v o u s s y s t e m . A s the conduction velocity did not show any c h a n g e s a single, although high dose of DDVP, s e e m e d to be insufficient to s e v e r e l y d a m a g e the conduction in p e r i p h e r a l n e r v e s . The increase of the absolute and relative r e f r a c t o r y periods, h o w e v e r , points to a subtle a l t e r a t i o n of its function. The d a t a of t h e subchronic e x p e r i m e n t s p r o v e d t h a t r e l a t i v e l y small doses of D D V P e x e r t e d a d e t e r i o r a t i n g effect on t h e functions of the central and p e r i p h e r a l n e r v o u s s y s t e m e v e n a f t e r a s h o r t period. I t is v e r y i m p o r t a n t , t h a t w i t h o u t a n y clinical s y m p t o m s of intoxication t h e functional p a r a m e t e r s of the central and p e r i p h e r a l n e r v o u s s y s t e m s , i.e. E E G m e a n frequency, E E G index, conduction velocity, r e f r a c t o r y periods w e r e altered. It is n o t e w o r t h y too, t h a t t h e r e w e r e no direct correlations b e t w e e n the obtained a l t e r a t i o n s of t h e c e n t r a l and p e r i p h e r a l n e r v o u s s y s t e m on t h e one hand and c h a n g e s of t h e c h o l i n e s t e r a s e activity of blood and o t h e r tissues on the o t h e r hand. W e believe t h a t our m e t h o d s d e m o n s t r a t e d here would be suitable to indicate e a r l y and mild c h a n g e s of t h e sensitive functions of the o r g a n i s m and t h u s will be applicable for t h e p r e v e n t i o n of p e r m a n e n t d a m a g e . REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12
148
W.A. Aldridge and M.K. Johnson, Side effects of organophosphorous compounds: delayed neurotoxicity. WHO Bull., 44 (1971) 259. M.K. Johnson, The anomalous behavior of dimethyl phosphates in the biochemical test for delayed neurotoxicity. Arch. Toxicol., 41 (1978) 107. M.K. Johnson, Delayed neurotoxicity: do trichlorphon and/or dichlorvos cause delayed neurpathy in man or in test animals? Acta Pharmacol. Toxicol., Suppl. 5, 49 (1971) 87. S. Caroldi and M. Lotti, Delayed neurotoxicity caused by a single massive dose of diehlorvos to adult hens. Toxieol. Lett., 9 (1981) 157. B.M. Francis, P.L. Metcalf and L.G. Hansen, Toxicity of organophosphorus esters to laying hens after oral and dermal administration. J. Environ. Sci. Health, B20(1) (1985) 73. K.M. Hyce, J.C. CrandaU, K.E. Kortman, and W.K. McCoy, EEG, ECG and respiratory response to acute insecticide exposure. Bull. Environ. Contain. Toxicol., 19 (1978) 47. J.C. Hazelwood, G.E. Stefan and J.M. Bowen, Motor unit irritability in beagles before and after exposure to cholinesterase inhibitors. Am. J. Vet. Res., 41(6) (1979) 852. I. D6si, Judit Szlobodnyik, M~ria Karmos and ~gnes Strohmayer, Neurotoxicological investigation of a new Hungarian pesticide Toxurazine. Acta Physiol. Acad. Sci. Hung., 60 (1982) 1. T. Miyoshi and I. Goto, Serial in vivo determinations of nerve conduction velocity in rat tails. Electroeneephalogr. Clin. Neurophysiol., 35 (1973) 125. Elisabeth Anda, Gy. Dura, I. L6rincz, I. Vincze and Magdolna Kert~sz, The effect of CO2 on the central nervous system (in Hungarian), Eg6szs6gtudom~ny, 28 (1984) 270. K.H. Meinecke and H. Oettel, Mikromethode zur Bestimmung der Acethylcholinesterase Aktivit~t in Erythrocyten und Plasma von Menseh und Tier. Arch. Toxicol., 22 (19671244. A.T. Modak, W.B. Stavihona and S.T. Weintraub, Dichlorvos and the cholinergic system: effects on cholinesterase and acetylcholine and choline contents of rat tissues. Arch. Int. Pharmacodyn. Ther., 217 (1975) 293.