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Skeletal muscle necrosis secondary to parathion
TOXICOLOGY
AND APPLIED
Skeletal
PHARMACOLOGY
Muscle
25, 117-l 22 (1973)
Necrosis WILLIAM
Department
Secondary B. KIBLER~
of Neurology,
Vanderbilt University Nashville, Tennessee 37232 Received
to Parathion192
July
School
of Medicine,
14, 1972
Skeletal Muscle Necrosis Secondary to Parathion. KIBLER, W. B. (1973). Toxicol. Appl. Pharmacol, 25, 117-122. Twenty-six Sprague-Dawley rats
were given daily ip injections of parathion for 2 weeks and sacrificed at regular intervals. The parathion induced histological evidence of skeletal muscle necrosis similar to lesions produced by paraoxon. The skeletal muscle necrosis is believed to be due to excessiveacetylcholine. Adaptation is rapidly developed, this may be the result of progressive refractoriness of the end plate. The organophosphate insecticide parathion has known toxic effects in man as a result of irreversible cholinesterase inhibition (DuBois ef al., 1949; Holmstedt, 1959). The clinical picture of the acute intoxication is a cholinergic crisis with major manifestations in both the central and peripheral nervous system (Durham and Hayes, 1962; Hayes, 1965). Reports of chronic intoxication are uncommon (Davignon et al., 1965) but the major disturbances are in neurological function (miosis, hypo or areflexia, tremor, weakness, and disturbance of equilibrium) and are probably the result of chronic low grade disturbances in cholinergic neurotransmitter systems. These studies do not report neuropathy and/or myopathy as a visible part of the clinical syndrome. Ariens (Ariens et u!., 1968, 1969) was the first to show that the single injection of paraoxon, the active metabolite of parathion, produced necrosis in skeletal muscles. More recently, Fenichel et uZ. (1972) demonstrated that the chronic administration of paraoxon in doses of about two-thirds the LD50 produces a progressive myopathy. The myopathy is prevented by denervation and modified by hemicholinium and is thought to be the result of excessive acetylcholine (ACh) stimulation at the neuromuscular junction. These findings raised the possibility that muscle fiber necrosis may be present and contribute to the clinical picture of parathion poisoning. The present study reports a histological study of skeletal muscle in rats chronically poisoned with parathion. METHODS Thirty-two white female Sprague-Dawley rats weighing 150-200 g were used. Six died owing to parathion toxicity; 26 were sacrificed by ip injection of pentobarbital sodium and studied. 1 From the Neuromuscular Disease Research Center (USPHS 1 POl-NS 10175), Department of Neurology. z Reprint requests to Dr. Gerald M. Fenichel, Department of Neurology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232. 3 United Cerebral Palsy Fellow. Copyright 0 1973 by Academic Press, Inc. 117 AI1 rights of reproduction in any form resewed.
118
KIBLER
Puruthion injection. Thirteen animals received daily ip injections of parathion at a dose of I mg/kg and 13 received 1.3 mg/kg. These doses represent about 0.5 to 0.75 the LD50. Animals receiving both doses were sacrificed on days 1,3, 5,8, lo,12 and 14 after the start of injections. Preparation of tissue. The quadriceps, gastrocnemius and soleus muscles were removed immediately after sacrifice in each animal. Portions of each muscle were rapidly
FIG. I. Phase I: Focal necrosis in thecentral portion of the muscle fiber following 5 days of parathion injections, 1 mg/kg. (DPNH. x 1280).
frozen in isopentane cooled in liquid nitrogen. Sections were prepared in a cryostat at a thickness of 10 p and further processed by the techniques for hematoxyhn and eosin (H&E), trichome, myofibrillar adenosine triphosphatase incubated at pH 10.4 (ATPase), DPN-linked lactate dehydrogenase (LDH) and reduced diphosphopyridine nucleotide dehydrogenase (DPNH). Study of tissue. To quantitate the appearance and progression of the myopathy, 2 low power fields with the largest number of abnormal fibers from each H&E and trichrome slide were selected for study. Each low power field contained about 1000
PARATHION
AND
MUSCLE
NECROSIS
119
fibers. The abnormalities were classified according to three phases : Phase I was characterized by focal areas of basophilia and disruption of the lacy intracellular architecture of the individual muscle fibers (Fig. 1); Phase II showed a progression to generalized cellular architectural disruption (Fig. 2); and phase III was characterized by complete disruption and phagocytosis of the muscle fibers (Fig. 3). The total number of each type of lesion was calculated per rat, per day.
FIG. 2. Phase II: The early focal necrosis progresses to a generalized fiber necrosis. Specimen from a rat receiving parathion for 6 days, 1 mg/kg. Trichrome x 1280.
RESULTS All the rats demonstrated several signs of acute parathion intoxication 20-30 min after each injection. These included generalized tremor, respiratory difficulty with tachypnea and mild cyanosis, lethargy, and decreased consciousness. These signs abated within 2 to 3 hr although the general level of activity remained depressed. There was some evidence of skeletal muscle weakness between day 5 and day 9. After 10 days, the signs of poisoning became much less marked, recovery after injection was more rapid, and residual effects were not seen between injections.
120
KIBLER
The animals receiving 1 mg/kg of parathion per day developed a progressive myopathy which reached a peak on day 8, after which the number of new lesions diminished (Fig. 4). Animals receiving 1.3 mg/kg developed large numbers of phase III fibers by the third day; while the appearance of new lesions progressed to day 5, these fibers did not continue to undergo degeneration and subsequent phagocytosis (Fig. 5).
FIG. 3. Phase III: The abnormal fibers are infiltrated by inflammatory cells and phagocytosis occurs. Specimen from a rat receiving parathion for 7 days, 1 mg/kg. Trichrome x 1280. DISCUSSION
The histopathology of the muscle lesions produced by chronic parathion administration is identical to the abnormality described after repeated injections of paraoxon (Fenichel et uZ., 1972). It is postulated that the parathion lesions, like the paraoxon lesions, are due to excessive ACh at the neuromuscular junction caused by the metabolism of parathion to paraoxon in vivo. These results are significant in that primary muscle damage resulting from chronic administration of moderate doses of parathion has not been previously reported. If this animal work can be extrapolated to human toxicology, the following points should
PARATHION
AND
MUSCLE
121
NECROSIS
be emphasized. At worst, less than 5 % of fibers are affected. This percentage is far too small to produce clinical weakness. There is the definite development of an adaptation to the reduced content of chohnesterase which prevents the myopathy from progressing after a few days. The mechanism of the adaptation is not fully known but may be due to the development of a refractoriness of the motor end-plate to ACh as a result of prolonged stimulation (Brodeur and DuBois, 1964). This end-plate insensitivity may contribute to the decreased work tolerance and hyporeflexia seen in workers with longterm exposure to parathion (Davignon et al., 1965).
AVERAGE OF GASTROCNEMIUS
2 gL
QUADRICEPS, AND SOLEUS
l7I6 I5 I4 13 l2II-
0
I
2 3 4 DAYS
5 6
7
OF PARATHION
8
9
IO II
I2
I3
I4
f I mg/kgI
FIG. 4. Average number of abnormal fibers in quadriceps, gastrocnemius, and soleus muscles induced by parathion in rats, 1 mg/kg/day. Phase I, central necrosis; Phase II, generalized necrosis; and Phase III, phagccytosis.
The skeletal muscle necrosis may also be significant in terms of acute parathion poisoning. After exposure to a single high dose, skeletal muscle necrosis appears rather rapidly and may involve the diaphragm and intercostal muscles. This could contribute to the respiratory paralysis, which is a major cause of mortality in acute poisoning. The incidence of acute and chronic parathion poisonings will be increasing as the organic phosphate compounds continue to replace DDT and chlorinated hydrocarbons as common insecticides. Paraoxon, phospholene (W. Kibler and G. Fenichel, unpublished observation), and probably all cholinesterase inhibitors seem capable of producing skeletal necrosis by allowing excessive ACh stimulation of muscle.
122
KIBLER
AVERAGE OF GASTROCNEMIUS
QUADRICEPS, AND SOLEUS
22.4 20 I9 l6l7I6 2 E i E : iL
l5I4 I3 izII IO St6-
ii
7-
$
6-
z a
54-
* :b,,,,,,,,\, ,,= 0
I
2
DAYS
3
4
5
6
7
OF PARATHION
9
9
IO II
I2
I3 I4
l l,3mg/kgl
FIG. 5. Average number of abnormal fibers in quadriceps, gastrocnemius, and soleus muscles induced by parathion in rats, 1.3 mg/kg/day. Phase I, central necrosis; Phase II, generalized necrosis; and Phase III, phagocytosis.
REFERENCES A. TH., COHEN, E. M., MEETER, E. AND WOLTHUIS, 0. L. (1968). Reversible necrosis in striated muscle fibers of the rat after severe intoxication with various cholinesterase inhibitors. Znd. Med. Surg. 37, 845-847. ARIENS, A. TH., MEETER, E., WOLTHUIS, 0. L. AND VAN BENTHEM, R. M. J. (1969). Reversible necrosis at the end-plate region in striated muscles of the rat poisoned with cholinesterase inhibitors. Experientia 25, 57-59. BRODEUR, J. AND DUBOIS, K. P. (1964). Studies on the mechanism of acquired tolerance by rats to O,O-diethyl S-2-(ethylthio) ethyl phosphorodithioate (Di-syston). Arch. Znt. Pharmacodyn. 149, 56S570. DAVIGNON, L. F., ST. PIERRE, J., CHAREST, G. AND TOURNAGEAN, F. J. (1965). A study of the chronic effects of insecticides in man. Can. Med. Ass. J. 92,597-602. DUBOIS, K. P., DOULL, J., SALERNO, P. R. AND Coon, J. M. (1949). Studies on the toxicity and mechanism of action ofp-nitropheryldiethyl-triophosphate (parathion). J. Pharmacol. Exp. Ther. 95,79-92. DURHAM, W. F. AND HAYES, W. J., JR. (1962). Organic phosphorus poisoning and its therapy. Arch. Environ. Health 5,21&47. FENICHEL, G. M., KIBLER, W. B., OLSON, W. H. AND DETTBARN, WOLF-D. (1972). Chronic inhibition of cholinesterase as a cause of myopathy. Neurology 22, 10261033. HAYES, W. J., JR. (1965). Parathion poisoning and its treatment. J. Amer. Med. Ass. 192,49-50. HOLMSTEDT, B. (1959). Pharmacology of organophosphorus cholinesterase inhibitors. Pharmacol. Rev. 11,567-588. ARIENS,
AND APPLIED
Skeletal
PHARMACOLOGY
Muscle
25, 117-l 22 (1973)
Necrosis WILLIAM
Department
Secondary B. KIBLER~
of Neurology,
Vanderbilt University Nashville, Tennessee 37232 Received
to Parathion192
July
School
of Medicine,
14, 1972
Skeletal Muscle Necrosis Secondary to Parathion. KIBLER, W. B. (1973). Toxicol. Appl. Pharmacol, 25, 117-122. Twenty-six Sprague-Dawley rats
were given daily ip injections of parathion for 2 weeks and sacrificed at regular intervals. The parathion induced histological evidence of skeletal muscle necrosis similar to lesions produced by paraoxon. The skeletal muscle necrosis is believed to be due to excessiveacetylcholine. Adaptation is rapidly developed, this may be the result of progressive refractoriness of the end plate. The organophosphate insecticide parathion has known toxic effects in man as a result of irreversible cholinesterase inhibition (DuBois ef al., 1949; Holmstedt, 1959). The clinical picture of the acute intoxication is a cholinergic crisis with major manifestations in both the central and peripheral nervous system (Durham and Hayes, 1962; Hayes, 1965). Reports of chronic intoxication are uncommon (Davignon et al., 1965) but the major disturbances are in neurological function (miosis, hypo or areflexia, tremor, weakness, and disturbance of equilibrium) and are probably the result of chronic low grade disturbances in cholinergic neurotransmitter systems. These studies do not report neuropathy and/or myopathy as a visible part of the clinical syndrome. Ariens (Ariens et u!., 1968, 1969) was the first to show that the single injection of paraoxon, the active metabolite of parathion, produced necrosis in skeletal muscles. More recently, Fenichel et uZ. (1972) demonstrated that the chronic administration of paraoxon in doses of about two-thirds the LD50 produces a progressive myopathy. The myopathy is prevented by denervation and modified by hemicholinium and is thought to be the result of excessive acetylcholine (ACh) stimulation at the neuromuscular junction. These findings raised the possibility that muscle fiber necrosis may be present and contribute to the clinical picture of parathion poisoning. The present study reports a histological study of skeletal muscle in rats chronically poisoned with parathion. METHODS Thirty-two white female Sprague-Dawley rats weighing 150-200 g were used. Six died owing to parathion toxicity; 26 were sacrificed by ip injection of pentobarbital sodium and studied. 1 From the Neuromuscular Disease Research Center (USPHS 1 POl-NS 10175), Department of Neurology. z Reprint requests to Dr. Gerald M. Fenichel, Department of Neurology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232. 3 United Cerebral Palsy Fellow. Copyright 0 1973 by Academic Press, Inc. 117 AI1 rights of reproduction in any form resewed.
118
KIBLER
Puruthion injection. Thirteen animals received daily ip injections of parathion at a dose of I mg/kg and 13 received 1.3 mg/kg. These doses represent about 0.5 to 0.75 the LD50. Animals receiving both doses were sacrificed on days 1,3, 5,8, lo,12 and 14 after the start of injections. Preparation of tissue. The quadriceps, gastrocnemius and soleus muscles were removed immediately after sacrifice in each animal. Portions of each muscle were rapidly
FIG. I. Phase I: Focal necrosis in thecentral portion of the muscle fiber following 5 days of parathion injections, 1 mg/kg. (DPNH. x 1280).
frozen in isopentane cooled in liquid nitrogen. Sections were prepared in a cryostat at a thickness of 10 p and further processed by the techniques for hematoxyhn and eosin (H&E), trichome, myofibrillar adenosine triphosphatase incubated at pH 10.4 (ATPase), DPN-linked lactate dehydrogenase (LDH) and reduced diphosphopyridine nucleotide dehydrogenase (DPNH). Study of tissue. To quantitate the appearance and progression of the myopathy, 2 low power fields with the largest number of abnormal fibers from each H&E and trichrome slide were selected for study. Each low power field contained about 1000
PARATHION
AND
MUSCLE
NECROSIS
119
fibers. The abnormalities were classified according to three phases : Phase I was characterized by focal areas of basophilia and disruption of the lacy intracellular architecture of the individual muscle fibers (Fig. 1); Phase II showed a progression to generalized cellular architectural disruption (Fig. 2); and phase III was characterized by complete disruption and phagocytosis of the muscle fibers (Fig. 3). The total number of each type of lesion was calculated per rat, per day.
FIG. 2. Phase II: The early focal necrosis progresses to a generalized fiber necrosis. Specimen from a rat receiving parathion for 6 days, 1 mg/kg. Trichrome x 1280.
RESULTS All the rats demonstrated several signs of acute parathion intoxication 20-30 min after each injection. These included generalized tremor, respiratory difficulty with tachypnea and mild cyanosis, lethargy, and decreased consciousness. These signs abated within 2 to 3 hr although the general level of activity remained depressed. There was some evidence of skeletal muscle weakness between day 5 and day 9. After 10 days, the signs of poisoning became much less marked, recovery after injection was more rapid, and residual effects were not seen between injections.
120
KIBLER
The animals receiving 1 mg/kg of parathion per day developed a progressive myopathy which reached a peak on day 8, after which the number of new lesions diminished (Fig. 4). Animals receiving 1.3 mg/kg developed large numbers of phase III fibers by the third day; while the appearance of new lesions progressed to day 5, these fibers did not continue to undergo degeneration and subsequent phagocytosis (Fig. 5).
FIG. 3. Phase III: The abnormal fibers are infiltrated by inflammatory cells and phagocytosis occurs. Specimen from a rat receiving parathion for 7 days, 1 mg/kg. Trichrome x 1280. DISCUSSION
The histopathology of the muscle lesions produced by chronic parathion administration is identical to the abnormality described after repeated injections of paraoxon (Fenichel et uZ., 1972). It is postulated that the parathion lesions, like the paraoxon lesions, are due to excessive ACh at the neuromuscular junction caused by the metabolism of parathion to paraoxon in vivo. These results are significant in that primary muscle damage resulting from chronic administration of moderate doses of parathion has not been previously reported. If this animal work can be extrapolated to human toxicology, the following points should
PARATHION
AND
MUSCLE
121
NECROSIS
be emphasized. At worst, less than 5 % of fibers are affected. This percentage is far too small to produce clinical weakness. There is the definite development of an adaptation to the reduced content of chohnesterase which prevents the myopathy from progressing after a few days. The mechanism of the adaptation is not fully known but may be due to the development of a refractoriness of the motor end-plate to ACh as a result of prolonged stimulation (Brodeur and DuBois, 1964). This end-plate insensitivity may contribute to the decreased work tolerance and hyporeflexia seen in workers with longterm exposure to parathion (Davignon et al., 1965).
AVERAGE OF GASTROCNEMIUS
2 gL
QUADRICEPS, AND SOLEUS
l7I6 I5 I4 13 l2II-
0
I
2 3 4 DAYS
5 6
7
OF PARATHION
8
9
IO II
I2
I3
I4
f I mg/kgI
FIG. 4. Average number of abnormal fibers in quadriceps, gastrocnemius, and soleus muscles induced by parathion in rats, 1 mg/kg/day. Phase I, central necrosis; Phase II, generalized necrosis; and Phase III, phagccytosis.
The skeletal muscle necrosis may also be significant in terms of acute parathion poisoning. After exposure to a single high dose, skeletal muscle necrosis appears rather rapidly and may involve the diaphragm and intercostal muscles. This could contribute to the respiratory paralysis, which is a major cause of mortality in acute poisoning. The incidence of acute and chronic parathion poisonings will be increasing as the organic phosphate compounds continue to replace DDT and chlorinated hydrocarbons as common insecticides. Paraoxon, phospholene (W. Kibler and G. Fenichel, unpublished observation), and probably all cholinesterase inhibitors seem capable of producing skeletal necrosis by allowing excessive ACh stimulation of muscle.
122
KIBLER
AVERAGE OF GASTROCNEMIUS
QUADRICEPS, AND SOLEUS
22.4 20 I9 l6l7I6 2 E i E : iL
l5I4 I3 izII IO St6-
ii
7-
$
6-
z a
54-
* :b,,,,,,,,\, ,,= 0
I
2
DAYS
3
4
5
6
7
OF PARATHION
9
9
IO II
I2
I3 I4
l l,3mg/kgl
FIG. 5. Average number of abnormal fibers in quadriceps, gastrocnemius, and soleus muscles induced by parathion in rats, 1.3 mg/kg/day. Phase I, central necrosis; Phase II, generalized necrosis; and Phase III, phagocytosis.
REFERENCES A. TH., COHEN, E. M., MEETER, E. AND WOLTHUIS, 0. L. (1968). Reversible necrosis in striated muscle fibers of the rat after severe intoxication with various cholinesterase inhibitors. Znd. Med. Surg. 37, 845-847. ARIENS, A. TH., MEETER, E., WOLTHUIS, 0. L. AND VAN BENTHEM, R. M. J. (1969). Reversible necrosis at the end-plate region in striated muscles of the rat poisoned with cholinesterase inhibitors. Experientia 25, 57-59. BRODEUR, J. AND DUBOIS, K. P. (1964). Studies on the mechanism of acquired tolerance by rats to O,O-diethyl S-2-(ethylthio) ethyl phosphorodithioate (Di-syston). Arch. Znt. Pharmacodyn. 149, 56S570. DAVIGNON, L. F., ST. PIERRE, J., CHAREST, G. AND TOURNAGEAN, F. J. (1965). A study of the chronic effects of insecticides in man. Can. Med. Ass. J. 92,597-602. DUBOIS, K. P., DOULL, J., SALERNO, P. R. AND Coon, J. M. (1949). Studies on the toxicity and mechanism of action ofp-nitropheryldiethyl-triophosphate (parathion). J. Pharmacol. Exp. Ther. 95,79-92. DURHAM, W. F. AND HAYES, W. J., JR. (1962). Organic phosphorus poisoning and its therapy. Arch. Environ. Health 5,21&47. FENICHEL, G. M., KIBLER, W. B., OLSON, W. H. AND DETTBARN, WOLF-D. (1972). Chronic inhibition of cholinesterase as a cause of myopathy. Neurology 22, 10261033. HAYES, W. J., JR. (1965). Parathion poisoning and its treatment. J. Amer. Med. Ass. 192,49-50. HOLMSTEDT, B. (1959). Pharmacology of organophosphorus cholinesterase inhibitors. Pharmacol. Rev. 11,567-588. ARIENS,