Delayed neurotoxicity from continuous low-dose oral administration of leptophos to hens

TOXICOLOGY AND APPLIED PHARMACOLOGY 38,595-608 (1976)

Delayed Neurotoxicity from Continuous Low-Dose Oral

Administration of Leptophos to Hens’

MOHAMED B. ABOU-DONIA AND SANDRA H. PREISSIG~

Department of Physiology and Pharmacology, and Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710

Received May 17,1976; accepted July 28, I976

Delayed Neurotoxicity from Continuous Low-Dose Oral Administration of Leptophos to Hens. ABOU-DONIA, M. B., AND PREISSIG, S. H. (1976). Toxicol. Appl. Pharmacol. 38, 595-608. Delayed neurotoxicity in hens was produced following daily oral administration of subneurotoxic doses, i.e., 1, 2.5, 5, 10, and 20 mg/kg of leptophos [0-(4-bromo-2,5-dichlorophenyl) O-methyl phenylphosphonothioate] in gelatin capsules for 60 days or until ataxia developed. A “no-effect” dose was found to be 0.5 mgfkg. Severity of the clinical condition depended on the size of the daily ingested dose, i.e., while paralysis was permanent after large doses, it was temporary after smaller doses. Duration of administration and “total dose” before onset of ataxia depended on the daily dose. The effect was not only cumulative but the total dose required to produce ataxia decreased with decrease of the daily dose. Degeneration of myelin and axons was seen in spinal cords and sciatic nerves of intoxicated birds. The extent of change varied with in- creasing dose. Paralyzed hens had significant histologic alteration while ataxic birds exhibited only mild changes. Red blood cell acetylcholinesterase was inhibited and recovered when the administration of leptophos had stopped. On the other hand, plasma cholinesterase, after initial inhibition and recovery, was severely inhibited as the signs of neurotoxicity progressed.

Leptophos (Phosvel) is a new phosphonothioate insecticide that is currently being considered in the United States for use on a variety of crops. A single oral dose of leptophos (Fig. 1) produces delayed neurotoxicity in hens similar to that reported for other

of leptophos.

I A preliminary account of this work has been presented (Abou-Donia and Preissig, 1975b). Z Present address: Department of Pathology, Health Science Center at San Antonio, The University

of Texas, 7703 Floyd Curl Drive, San Antonio, Texas 78284. Copyright 0 1976 by Academic Press, Inc. 595 All rights of reproduction in any form reserved. Printed in Great Britain

596 ABOU-DONIA AND PREISSIG

neurotoxic organophosphorus compounds (Abou-Donia et al., 1974; Abou-Donia and Preissig, 1975a, 1976; Abou-Donia, 1976a). Unlike most of the other organophosphorus pesticides, leptophos is relatively persistent in the environment (Leuck et al., 1969,197O; Johnson et al., 1971; Aharonson and Ben-Aziz, 1974); e.g., leaves of cotton plants still retain 29 % 9 weeks after application (Holmstead et al., 1973). Leptophos is very persistent in tissues of hens fed [14C]phenyl leptophos (Abou-Donia, 1976a,b,c).

It has been suggested from cases of human poisoning that small subneurotoxic doses could build up to cause neurotoxic effects (Birdstrup et al., 1953). This was found to be true when daily feeding of small doses of tri-o-cresyl phosphate (TOCP) to hens caused paralysis (Smith et al., 1932), and a no effect dose was determined to be 2.5 mg/kg. Similar cumulative effects with TOCP (Cavanagh, 1973, Henschler, 1958), diisopropyl phosphorofluoridate (DFP) (Davies and Holland, 1972), tri-p-ethyl phenylphosphate and mono-o-propyl di-p-ethyl phenylphosphate (Cavanagh, 1973) were obtained later.

This report gives an account of the investigation into the effect of continuous daily oral administration of small doses of leptophos to hens.

METHODS

Leptophos [O-(4-bromo-2,5-dichlorophenyl) O-methyl phenylphosphonothioate] technical grade (94.8 % ir, 87.2 % gc) was provided by Velsicol Chemical Company, Chicago, Illinois.

Care and treatment of birds. Laying hens (Gallus gallus domesticus), mixed breed, each 19 months old and weighing approximately 2 kg (1.4-2.2 kg) were used. The birds were placed in individual cages in an air-conditioned room and allowed to adjust to the environment for 1 week before the beginning of the experiment. Six groups of birds (three birds each) were given daily a single oral dose of 0.5, 1,2.5, 5, 10, and 20 mg/kg of technical leptophos. Three hens were given empty gelatin capsules and served as controls (Table 1). The oral administration continued daily until ataxia developed, but no longer than 60 days. The birds were returned to their cages and supplied with food3 and water ad libitum. All birds were examined daily in order to detect any abnormality in gait or behavior. Body weights were monitored daily during the experiment. Eggs werecollected and weighed. The birds died or were sacrificed after they developed marked signs of paralysis and their conditions deteriorated. Blood samples were taken at varying intervals of the experiment.

Histological methods. Brains, spinal cords, and sciatic nerves of hens were excised and prepared for histological examination as described before (Abou-Donia and Preissig, 1976).

Enzymatic analysis. Red blood cell acetylcholinesterase (AChE) and plasma cholinesterase (ChE) activity were measured by methods described previously (Abou-Donia and Menzel, 1967; Abou-Donia and Preissig, 1976). Heparinized, freshly drawn blood was centrifuged, the plasma was removed, and the red cells were washed twice with 0.9 % solution of sodium chloride. RBC-AChE measurements were made by observing acetylthiocholine (ATCH) hydrolysis in a Varian Techtron Model 635 UV-Vis spectro- photometer at 412 nm using 5,5’-dithio-bis-2nitrobenzoic acid (DTNB) (Ellman et al., 1961). Plasma ChE was measured by observing the hydrolysis of butyrylthiocholine

3 Layena chicken feed, Ralston Purina Company, St. Louis, Missouri.

NEUROTOXICITY OF LEPTOPHOS 597

(BUTCH) at 412 nm using DTNB. The reactions were carried out at 38°C in a Dubnoff metabolic shaker for 5 min. Parallel blank incubations were carried out, utilizing heat- denatured RBC or plasma solutions for each determination. RBC and plasma proteins were determined by the method of Lowry et al. (1951). The results are expressed as micromoles ofATCH hydrolyzed per minute per milligram of protein, and as nanomoles of BUTCH hydrolyzed per minute per milligram of protein for RBC-AChE and plasma ChE, respectively.

TABLE 1

EFFECT ON NUMBER AND WEIGHT OF EGGS LAID FOLLOWING THE DAILY ADMINISTRATION OF A SINGLE ORAL DOSE OF LEPTOPHOS TO HENS

Hen Weight Dose Eggs per hen Egg weight Last day of

(kg) (wdk) per day (mean + SE g) laying

A B C D E F G H I J K L M N 0

t R

Control 1 Control 2 Control 3

1.86 20.00” 2.14 20.00 1.97 20.00” 1.73 10.00 1.90 10.00 2.07 10.00 1.85 5.00 2.23 5.00” 2.22 5.00” 2.42 2.50 1.51 2.50 2.00 2.50 1.59 1.00 1.50 1.00 1.43 1 .oo 1.97 0.50 1.77 0.50 1.97 0.50 1.45 0.00 2.40 0.00 1.95 0.00

0.07 43.68 + 2.16” 0.37 48.89 f 1.23c 0.28 53.92 k 2.24’

b b

0.14 63.38 + 0.57 b b

0.12 0.14

b

0.34 0.41 0,78 0.78 0.49 0.72 0.49 0.40 0.74 0.75 0.76 0.77

58.89 + 1 .23d 60.46

b

54.00 + 1 .70d 59.58 + 2.86d 61.20 + 1 .70d 66.13 f 0.45 55.08 + 0.36d 60.41 + 0.31d 69.48 &- 1.04 63.01 f 0.92 67.36 of: 0.71 65.60 k 1.67 66.43 f 1.32 65.81 + 1.65

28 19 21 -

214 -

213 7

- 101 101 101 158 158 157 77 78 78

216 215 215

u Hens died shortly before dissection. ’ No eggs were laid. c Significant difference froni control (p < 0.001). d Significant difference from control (p < 0.02).

Statistics. Significance ofthe difference between control and treated birds was assessed by Student’s t test, and ap value of 0.05 or less was considered significant.

Body Weight

RESULTS

Continuous oral administration of leptophos caused weight loss in hens treated with all doses except 0.5 mg/kg (data not shown). The loss of weight was dose dependent. In


general, weight loss was high after paralysis developed. Some hens, however, showed increased body weight as their clinical conditions improved. Hens fed 0.5 mg/kg did not lose weight throughout the experiment.

Table 1 shows that daily oral administration of a small single oral dose of leptophos caused adverse effects on egg weight and production in all dose levels except hens given 0.5 mg/kg which were sacrificed at 78 days. Egg weights from all the treated hens except Hens E, M, P, Q, and R were significantly less than that from the controls.

Clinical Assessments

All hens given 20 mg/kg of leptophos developed ataxia which progressed to paralysis (Table 2). These hens showed all the signs of neurotoxicity reported for a large single oral dose of leptophos. Hens given 10 mg/kg developed ataxia and paralysis. These hens, however, recovered from paralysis about 2 months later. Although their clinical

TABLE 2

SEQUENCE OF INTOXICATION, ONSET OF CLINICAL SIGNS, AND HISTOLOGICAL CHANGES IN TISSUES FROM HENS GIVEN A DAILY SINGLE DOSE OF LEPTOPHOS~

Hen Dose

Days of administration Histological changes

Paralysis Duration of Sciatic Spinal

Ataxia Onset Recovery Sacrifice intoxicationb nerve cord

A 20.00 29 39 - 85” 56 + ++ B 20.00 22 26 - 50 28 + ++ C 20.00 29 35 - 77 48 NE NE D 10.00 39 58 102 108 69 - + E 10.00 39 56 112 216 177 - + F 10.00 32 61 102 216 184 - + G 5.00 59 - - 216 157 - +i-- H 5.00 58 - - 59’ 1 NE NE I 5.00 59 - - 123c 64 - I!I J 2.50 25 - - 103 78 +I- +/- K 2.50 65 - - 103 38 - - L 2.50 65 - - 103 38 NE NE M 1.00 68 - - 160 92 NE NE N 1.00 62 - 0 1.00 62 68 63

160 98 NE NE 160 98 - -

P 0.50 - - - 78 - NE NE Q 0.50 - - - 78 - NE NE R 0.50 - - - 78 - - -

0 The following abbreviations are used: NE, tissues not examined; -, changes absent; +/-, changes equivocal ; +, changes present ; ++, severe degeneration.

b Interval between onset of ataxia and sacrifice. c Hen died shortly before dissection.

NEUROTOXICITY OF LEPTOPHOS

D-

3-

D-

o-

o- *

I 0 5 IO 15 20

Daily Dose (mg/Kg)

FIG. 2. Effect of a daily oral dose of leptophos on the period of time before onset of ataxia.

599

Daily Dose (mg/Kg)

FJG. 3. Effect of a daily single oral dose of leptophos on the total dose before ataxia developed in hens.


conditions improved, they never completely recovered from ataxia. Hens given 5 and 2.5 mg/kg of leptophos developed ataxia but showed no paralysis. The clinical conditions of these hens improved, but recovery was not complete. The fifth group of hens was given a daily dose of 1 mg/kg of leptophos for 60 days. All birds developed ataxia. The condition of one of these hens progressed to paralysis, with subsequent recovery

FIG. 4. Cervical spinal cord of Hen G. The high power view of the lateral column of cervical cord shows a swollen and degenerated axon. I-3 &E-LB, x 250.

6 days after the oral administration of leptophos had stopped. The conditions of these hens improved with time. The last group of hens was given a daily dose of 0.5 mg/kg of leptophos for 60 days and was sacrificed at 78 days. These hens showed no abnormality in gait or behavior. The maximum dose which did not cause an effect is called the “no- effect” dosage. Figure 2 shows that duration of administration of subneurotoxic doses of leptophos before onset of ataxia depended on and was inversely proportional to the


size of the daily single dose. Thus, when a daily dose of 1 mg/kg was given to hens, 60 days of administration were required to produce ataxia as compared to an average of 27 days when 20 mg/kg was administered. Figure 3 shows that the “total dose” before ataxia developed differed and depended on the daily dose. Thus, when a daily dose of 20 mg/kg was orally administered the “total dose” given before onset of ataxia averaged 533.3 mg/kg as compared to 64.0 mg/kg when 1 mg/kg was given daily for 60 days.

Histopathological Changes

In some of the hens given 2.5 mg/kg, occasional swollen and fragmented sheaths and axons in the spinal cord (Table 2) were present. These changes involved isolated

FIG. 5. Thoracic spinal cord of Hen A. This cross section shows myelin loss in the anterior column of the white matter. H & E-LFB, x 25.

fibers but were generally in the same tracts damaged in animals fed higher doses. The latter hens showed swollen and degenerating myelin sheaths and axons involving the posterior and lateral columns in the cervical cord and the middle portion of the anterior columns in the lumbar cord (Figs. 4 and 5, and Table 2). In hens given high doses, the lateral portion of the medulla showed swelling and fragmentation of myelin and axons similar to changes in the spinal cord. Cerebellum, cerebrum, and optic lobes had no histopathological changes as compared to the controls.

The sciatic nerve divides into two main branches, the tibia1 and the peroneal, fairly


soon after leaving the pelvic region, which are bound together by a loose connective tissue sheath. Approximately two-thirds of the way down the thigh these separate into two distinct nerves. No histological changes were seen in the sciatic nerves of most of the treated hens as compared to nerves from control hens. The only unequivocal changes were seen in the severely poisoned hens which were given a daily 20-mg/kg dose of

FIG. 6. Sciatic nerve of Hen B. The clear vacules are swollen, disrupted axon and myelin sheaths. Masson, x 440.

leptophos (Table 2). These lesions were much more easily seen in tibia1 and peroneal nerves below the division into separate nerves. The changes consisted of numerous foci of swollen myelin sheath containing fragmented myelin and axons (Figs. 6 and 7). Holme’s silver stain showed disrupted axons.

Cholinesterases

Daily oral administration of a single dose of leptophos inhibited RBC-AChE (Fig. 8). In general, there was a dose-dependent inhibition of RBC-AChE. The inhibition was greatest on the last day of administration. After leptophos administration was stopped, there was a slow recovery in the enzymatic activity followed by a fast rate of increase. At the end of the experiment the activity of RBC-AChE from hens given 20 and 10 mg/ kg reached the control value. RBC-AChE from hens given 5 mg/kg did not show any recovery since they were fed leptophos up to 58 and 59 days.


Plasma ChE activity of treated birds was significantly reduced throughout the experiment (Fig. 9). Again, an increase in dose produced an increase in enzymatic inhibition. After the administration of leptophos had stopped, some recovery of plasma ChE activity was noted in hens given 10 and 20 mg/kg daily. A secondary depression

FIG. 7. Sciatic nerve of Hen B. This portion of sciatic nerve shows axonal disruption and prolifera- ting Schwann cell nuclei. Holme’s stain, x 440.

was noted in all treated birds. Plasma ChE activity values remained significantly lower than that of the control thereafter.

DISCUSSION

The introduction of organophosphorus insecticides displaying delayed neurotoxicity into our food supply and environment, adds a new dimension to safety considerations for the general public and farm workers. In the present investigation, the experimental insecticide leptophos was used as a model compound of this group of neurotoxic insecticides. This compound is relatively persistent in the environment and has a long


0 40

1.

Tb

8 20mg/“- R

2. c

0 IO mg/Kg C

. 5 mg/Kg

, I I I I 0 IO 20 30 40 50 60

Days

FIG. 8. Effect of a daily single oral dose of leptophos (5, 10, or 20 mg/kg) on RBC-AChE activity. The letters A to I refer to hens as designated in Table 2. The administration of the leptophos was stopped at times indicated by the arrows. Results are calculated as percentage of activity measured in red blood cells from normal birds taken at the same time. Each point represents the mean of two determinations from each of one to three birds. The mean and SE of 10 control rates was 30.5 + 2.75 pmol of ATCH hydrolyzed per minute per milligram of protein.

100 t

0 IO 20 30 40 50 60

Days

FIG. 9. Effect of a daily single oral dose of leptophos (5, 10, or 20 mg/kg) on plasma ChE activity. The letters A to I refer to hens as designated in Table 2. The administration of the leptophos was stopped at times indicated by the arrows. Results are calculated as percentage of activity measured in plasma from normal birds taken at the same time. Each point represents the mean of two determinations from each of one to three birds. The mean and SE of 10 controls was 1150 + 53 nmol of BUTCH hydrolyzed per minute per milligram of protein.


biologic half-life in hens fed [14C]phenyl leptophos (Abou-Donia, 1976a,b,c). It produces delayed neurotoxicity when orally administered to chickens in a single dose 25 times less than the lethal dose (Abou-Donia et al., 1974).

This study shows that the daily oral administration of small subneurotoxic doses of leptophos ranging between 1 and 20 mg/kg can build up and cause neurotoxic effects in hens. The dose of 1 mg/kg was a “threshold dose” below which no effects were produced. A “no-effect” daily oral dose of 0.5 mg/kg was found, which produced no signs of ataxia, no histological changes, and no marked variation in the weight and number of eggs laid after 60 days of oral administration. The duration of administration of subneurotoxic doses before ataxia developed varied inversely with the size of the daily dose. Also, the “total dose” before onset of ataxia was proportional to the size of the daily ingested dose. However, this was not merely a cumulative effect, since at lower daily doses there was a decrease in the total dose required to produce ataxia. Thus, one single dose of 160 mg/kg of leptophos was ineffective (Abou-Donia et al., 1974), while 60 daily doses of 1 mg/kg were neurotoxic. This finding may be attributed to a more efficient absorption and/or metabolism of divided doses than of a single dose. Leptophos could have an effect on absorption of itself, and probably of nutrients, as is shown by changes in body weights and in the levels of egg production. Myers and Mulder (1953) and Myers et al. (1955) have shown that tocopherol deficiency produced by TOCP in animals on a normal diet was probably the consequence of an interference with the absorption of Vitamin E in the intestine. Tocopherol deficiency could be a contributing factor to the irreversible sterility of male rats chronically fed small doses of TOCP (Mendel and Myers, 1952; Draper et al., 1952) and to the reduction in number and weight of eggs laid by hens given leptophos. This effect might also be caused directly by the esterase inhibition. It is not known to what extent the deficiency contributes to neuropathology. Majno and Karnovsky (1961) have reoorted that in the sciatic nerves of hens, respiration and especially the incorporation of acetate and phosphate into the total lipids were significantly depressed by nutritional deficiency. Samples of white matter from the spinal cord were not demonstrably affected.

The results of the present investigation are in accord with the finding that the oral administration of 20 to 30 daily doses of 5 mg/kg of TOCP to hens caused paralysis (Davies and Holland, 1972). However, when the daily dose was reduced to 2.5 mg/kg, no effect was produced after 76 doses. When doses were spread over 40 days, 50 to 100 % more TOCP was needed to produce ataxia (Henschler, 1958). Hens fed a daily diet containing 800 ppm of TOCP became ataxic by 16 days (Cavanagh, 1964a). However, at 100 ppm no clinical or histological changes could be found after 150 days of feeding. Divided doses of DFP showed some cumulative effect when fed to chickens, even when they were separated by an interval as long as 16 days (Davies and Holland, 1972).

The pattern of myelin and axon degeneration in spinal cords and sciatic nerves after continuous oral administration of small doses to hens was identical to that produced by a single dose of leptophos (Abou-Donia and Preissig, 1975a, 1976; Preissig and Abou-Donia, 1976) and to that found in the classical picture of organophosphorus compound neurotoxicity (Cavanagh, 1954, 1963, 1964b, 1973 ; Barnes and Denz, 1953; Lancaster, 1960; Beresford and Glees, 1963).

The severity of histologic changes did not correlate with the time between the onset of ataxia and sacrifice, but depended on the dose. Minimal alterations were seen in


other animals showing only ataxia. Severe changes were present in the chickens receiving 20 mg/kg. The extent of spinal cord lesion explains paralysis and makes recovery unlikely. The clinical improvement of the animals receiving 10 mg/kg is interesting. It is unlikely that this improvement represents regeneration within the spinal cord, as the repair phenomenon is not typical of the central nervous system (Young, 1942). On the other hand, it is possible that extensive peripheral nerve damage was repaired (Guth, 1956). Perhaps spinal cord changes of an acute and reversible nature, such as edema, were present and later subsided. Alternately, the initial paralysis might represent a significantly more profound effect at the molecular level than is represented by histological changes. The latter explanation seems to hold true for the animals given single doses of 5.0 and 2.5 mg/kg, which showed ataxia with minimal histologic alterations.

In the present investigation, after the daily oral administration of 10 and 20 mg/kg of leptophos had stopped, an activation of plasma ChE followed by a second depression was noted. These results are in agreement with the effect of DFP and Mipafox (N&V’- diisopropyl phosphorodiamidic fluoride) on pseudo ChE in the cervical spinal cord of hens (Davison, 1953). It has been shown that in man the plasma ChE activity is lowered in states of malnutrition (Hutchinson et al., 1951). A comparison of weight data (not shown) and data on Fig. 9, however, shows that reduction of plasma ChE activity began at a time when the hens had lost very little weight. Also, the brief enzyme recovery began at a time when the hens had lost more weight and were still losing it at an increased rate. Thus, the reason for the change in plasma ChE must be more specific than general malnutrition. The second depression of plasma ChE beginning at about the fortieth day, may have been influenced by the serious malnutrition of the chickens at that time. Observation on the plasma cholinesterase activity in the human cases of poisoning by Mipafox showed that the pseudocholinesterase remained at a low level for many weeks (Bidstrup et al., 1953).

It has been suggested that the initial event in the delayed neurotoxicity may be inhibition of an esterase (Koelle and Gilman, 1946; Aldridge, 1953). Subsequently, it has been shown that there is in brain an esterase, which hydrolyzes phenyl phenyl- acetate, a small portion of which (3 to 5 %) ( neurotoxic esterase) is phosphorylated by neurotoxic but not by nonneurotoxic organophosphorus compounds (Johnson, 1975a). Of interest is the finding that leptophos oxon [O-(4-bromo-2,5-dichlorophenyl) O- methyl phenylphosphonate] is a potent inhibitor of neurotoxic esterase of hen brain in vitro (Johnson, 1975b).

This communication has reported experiments showing that oral administration of small subneurotoxic doses of leptophos can build up to cause neurotoxic effects in hens. These results are in agreement with the effects of other neurotoxic organophosphorus compounds. Leptophos should be handled carefully, in view of its stability and the neurotoxic effects produced by consistent feeding of small doses of this insecticide.

ACKNOWLEDGMENTS

Acknowledgment is made to Ms. Jean L. Gerth and Mrs. Jacqueline H. Carothers for their technicaf assistance, and to Ms. Elaine Smolko for her secretarial work. The supply of technical leptophos by Velsicol Chemical Corporation (Chicago, Illinois) is acknowledged. This study was supported in part by NIH Fellowship No. 1 F22 ES01723-02, and EPA Contract No. 68-02-2452.


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Delayed neurotoxicity from continuous low-dose oral administration of leptophos to hens

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