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The acute toxicity of sarin in marmosets (Callithrix jacchus): A behavioral analysis
FUNDAMENTAL
AND
APPLIED
TOXICOLOGY
5,
S 169-S
174 (
1985)
The Acute Toxicity of Sarin in Marmosets (Callithrix jacchus): A Behavioral Analysis G. D. D'MELLO' AND E. A. M. DUFFY Chemical Defence Establishment, Porton Down, Salkbury, Wiltshire, United Kingdom SP4 OJQ
The Acute Toxicity of Satin in Marmosets (Callithrir jacchus): A Behavioral Analysis. D’MELLO, G. D., AND DUFFY, E. A. M. (1985). Fundam. Appl Toxicol. 5, Sl69-S 174. In marmosets, the clincial signs of poisoning following the administration of satin and the effects of satin upon performance in three behavioral test models were investigated. The sensitivity of marmosets to the lethal action of satin was shown to be greater than that of rodent species and rabbits but very similar to that of the rhesus monkey. Doses of sarin ranging from 33 to 55% LD50, resulting in erythrocyte acetylcholineatemse inhibitions of 88% or mom, were shown to disrupt the performance of a food-reinforced visually guided reaching response. However, the pattern of disruption did not suggest a specific action of satin upon visuomotor coordination and could not be explained on the basis of an indirect effect of this agent upon either feeding motivation or changes in gross mobility. It was suggested that possible changes in animals’ perception of the reaching task and/ or their own drugged ability may be relevant to any interpretation of satin-induced changes in visuomotor coordination in this species. Q 1985 Academic Pres, Inc.
Sublethal doses of organophosphorus compounds are known to produce symptoms likely to degrade normal function in humans (Brimblecombe, 1974). Research methods are therefore required to characterize those sublethal, and in particular the behavioral, effects of those organophosphorus compounds which are possible chemical warfare agents (Hamm, 1984). One traditional approach involves the selection of a rodent species such as the rat and a wide range of neurobehavioral test models (Tilson et al., 1980; Weiss and Laties, 1975). However, the relevance of rodent behavioral models for humans is debatable. Although the so-called higher primates are regarded generally as the species of choice, they are not readily available to the majority of experimenters. A requirement therefore exists for the development of neurobehavioral test methods in a primate species that is readily available.
’ To whom correspondence should be addressed.
The work to be described here represents part of a research program to characterize the behavioral effects of sarin in marmosets (Cullithrix jacchus), a primitive primate but one which is gaining popularity in behavioral pharmacological research (Campos and Arruda, 1981; D’Mello et al., 1985; Scraggs and Ridley, 1978). To facilitate interspecies comparison the lethal potency of sarin and the incidence and duration of signs of poisoning were determined. On the basis of symptoms observed in humans following mild poisoning by acetylcholine&erase (AChE) inhibitors (i.e., <50% erythrocyte AChE inhibition), (Ministry of Defence, 1972) and consideration of the effects of these agents upon neuromuscular function (Hobbiger, 1976), changes in voluntary motor output are expected. AChE inhibitors have been reported to disturb a variety of motor behaviors in both rodent (Fatehyab et al., 1980; Landauer and Romano, 1984; Wolthuis and Vanwersch, 1984) and primate (Brack and Rothe, 1982; Grob, 1963) species.
S169
0272-0590/85 $3.00 Copyright 8 1985 by the Sociely of Toxicology. All rights of reproduction in any form d.
s170
D’MELLO
However, the effects of these agents upon fine motor control have not been investigated fully. The influence of sarin upon a food-reinforced, visually guided reaching response has therefore been investigated using a method developed previously in this laboratory (D’Mello et al., 1985). Since performance on this task may be influenced by a number of behavioral components, notably feeding motivation and gross mobility, ancillary experiments were conducted to assessthese factors and also to provide a better understanding of those behavioral components mediating op timal performance on this task. A preliminary account of some of these data has been reported (D’Mello et al., 1983). METHODS Animnls. Marmosets of both sexesbred at the Chemical Defence Establishment were used. Each animal was housed individually in a cage (750 X 480 X 600 mm) located within a room maintained at 22-23°C and at a relative humidity of SO-60%, with a regular day-night cycle imposed by electric lighting (light from 7%) AM to 7:OOPM). All subjects were maintained at their free feeding body weights (range 290-380 g) on a high-protein diet (Mazuri primate diet, SDS, Witham, Essex, U.K.) with fruit and vitamin supplement. Apple and banana were withheld from those animals used in either the visually guided reaching or feeding motivation studies. Acute toxicity. Each animal was administered one of five doses of satin (range 15-25 &kg) and the signs of poisoning were monitored as a function of time up to 48 hr. No attempt was made to either quantify the intensity of the signs observed or to distinguish between various sign subtypes (e.g., clonic or tonic convulsions, resting or intentional tremors). At 48 hr, the number of deaths was recorded. Visually guided reaching. Marmosets were trained to retrieve small cylinders of apple from a moving belt positioned in front of their home cages. Access to the belt was restricted and belt velocity increased progressively during training to impose a maximum response time of less than 0.5 sec. Belt direction was changed on alternate trials, of which there were 50 each session. Responses were categorized according to four criteria. A “successful retrieval” was recorded if the animal caught and then ingested the pellet. A “failure” with or without contact was recorded when animals reached for but failed to retrieve the pellet. A “no-attempt” was recorded when animals did not respond. A detailed description of the apparatus and procedure has been reported (D’Mello et aZ., 1985).
AND DUFFY On the basis of data collected during the final week of training, five groups (n = 7 or 8) were selected, matched for performance, and allocated randomly to one of five treatments (saline or sarin 57.5, 10, or 12.5 pg&g). Testing was conducted on three consecutive days. On Day 1, all groups received saline. On Day 2, each group received their allocated treatment. On Day 3, all groups were tested for any residual treatment effects. Feeding motivation. Marmosets were trained to consume a fixed number (49 or 50) of apple pellets within a 5-min period. One group (n = 7) was presented with pellets in a feeding bowl placed within their home cages.Latency to begin feeding, the cumulative duration of feeding, and the number of pellets consumed were recorded. In a second group (n = 8) each animal was removed from its home cage and held within the gloved hands of an experimenter such that voluntary movements of the upper trunk, arms, and head were relatively unrestricted. A second experimenter presented pellets singly to the animal via a purposebuilt dispenser. Pellets were presented in close proximity to the animal’s mouth (2-3 cm) at a rate determined by the speed at which each animal was able to consume the pellets. Animals received a single feeding session each day until stable baseline levels of feeding were established. Testing was conducted on three consecutive days as described for those groups trained on the reaching task. The influence of a single dose level of satin (12.5 pg/kg) was assessed and corresponded to the highest dose of satin tested in the reaching task. The total number of pellets available and session time were selected to facilitate direct comparison with the reaching experiment. It was hypothesized that any changes in feeding motivation, as indicated by the number of pellets consumed, mediated indirectly by changes in gross mobility, would manifest in the “bowlfeeding” group but not in the “hand-feeding” group. Activity monitoring. The gross mobility or locomotor activity of groups of marmosets (n = 4) was monitored using a device developed previously to monitor activity levels in humans (Laporte et al. 1979). The device (37 X 30 X 13 mm; weight I9 g) was mounted on the back of an animal by means of a simple elastic harness. Movements by the animal in the anterorostral plane operated a simple mercury tilt switch, closures of which were used as activity counts. Counts were cumulated and stored by integrated circuitry and could be displayed in digital form following the manual operation of a separate magnetic switch. To monitor changes in the level of activity as a function oftime, the animal was caught by an experimenter at various times over a period of 4 hr and cumulated counts recorded. Testing was conducted within the home cage of each animal over three consecutive days. On Day 1, the device was fitted and each animal given 24 hr to habituate to its presence. On Day 2, control readings were taken. On Day 3, animals were injected with either saline or satin (7.5, 12.5, or 17.5 rcglkg), and readings were taken at times corresponding to those of Day 2.
BEHAVIORAL
TOXICITY
s171
OF SARIN IN MARMOSETS
Drug administration. Satin was synthesized by the Organic Chemistry Section at Chemical Defence Establishment. Dilutions were made with 0.9% saline solution. All injections were given intramuscularly in the inner thigh in a volume of 0.5 ml/kg body wt. In the visually guided reaching and feeding motivation tests, saline or sarin was administered 60 min before test sessions. This test time was selected to correspond with the time of peak erythrocyte AChE inhibition determined as described below. AChE activity. Blood samples were taken from the tail vein of separate groups of animals and then assayed for erythrocyte AChE activity using the spectrophotometric method of Ellman et al. (1961). Acetylthiocholine (1.0 mM) was used as the substrate. Statistical analysis. Statistical methods to determine group differences and appropriate to the nature of the data analyzed were selected according to the criteria described by Siegel (1956). Interval data (i.e., temporal scores) were first subjected to homogeneity of variance tests (F tests) and, if homogenous, compared using either paired or unpaired t tests asappropriate (Winer, 1971). For nonhomogenous data, groups were compared using either the Wilcoxon matched pairs, signed ranks test or the KolmogorovSmirnov two-sample test as appropriate (Siegel, 1956). Categorical data (i.e., frequency scores) were analyzed by means of x2 or Fisher’s exact probability tests(Siegel, 1956). LD50 values, based upon mortality data at 48 hr, were estimated using probit analysis (Finney, 197 1).
tion, and unsteadiness were observed in the majority of animals irrespective of dose. The first four of these signs were continuous in nature and were generally observed for periods in excess of 4 hr following poisoning. Bouts of increased mastication, swallowing, and nictitation were also observed frequently. There was a low incidence of vomiting (11%) and diarrhea (22% of animals tested). There were no overall differences in the frequency of signs observed between animals that received high (25 &kg) or low (15-20 pg/kg) doses of sarin. These doses corresponded to the estimated LD 100 and LD 10 doses for sarin, respectively. The incidence of vomiting and diarrhea seemed higher in those animals that received low doses, but this did not reach statistical significance. The first signs observed ca. 20 set following intramuscular injection of sarin were generally tremors, unsteadiness, or increased mastication. The times to onset of the first sign and of tremors and convulsions were significantly lower in animals that received the high dose of sarin (Table 1).
RESULTS Acute Toxicity
Visually Guided Reaching
The 48-hr intramuscular LD50 for sarin in marmosets was estimated to be 22.3 (17.923.6) &kg. Salivation, tremors, fasciculations, loss of posture, convulsions, piloerec-
Baseline levels of performance on this task for each of the five groups tested following saline administered on Day 1 did not differ. About 85% of total trials resulted in successful
TABLE 1 MEAN
TIME (MIN)
Dose of satin k&g)
TO ONSET OF FIRST OBSERVED AND MOST FREQUENT SIGNS FOLLOWING ADMINISTRATION OF SARIN IN MARMOSETS (N = 6 OR 9)
First observed sign
Salivation
15-20 (
1.9 (kO.6)
(,E)
(L&O)
(228;
(3)
Tremor
INTRAMUSCULAR
Convulsions 8.6 (e1.7)
,,i::;
(2::;
Note. Two animals in the 25 &kg dose group did not convulse. Numbers in parentheses show + 1 SE. Asterisks indicate significant difference between dose levels (p < 0.05).
S172
D’MELLO
AND DUFFY
retrievals. Animals responded but failed to retrieve on about 12% of trials. One hour following the injection of sarin, at doses resulting in erythrocyte AChE inhibitions of 88% or more, the number of successful retrievals was decreased in a dose-related manner (Table 2). At doses of satin between 7.5 and 12.5 &kg decreases in successes were accompanied by increases in no-attempts with little evidence for an increase in failures. Although signs of poisoning were not monitored routinely during this study, tremors and fasciculations were observed frequently both before and during testing. Twenty-four hours following the injection of sarin (Day 3), performance on the reaching task returned to predrug baseline levels in all groups (not shown). Feeding Motivation One hour following saline administration (Day 1) animals presented with 50 apple pellets in a bowl placed in their home cages began feeding within an average 2.4 set and consumed all pellets at an average rate of 20/min. One hour following administration of 12.5 & kg of satin on Day 2, these same animals took an average 208 set to begin feeding and con-
sumed a median of 20 pellets before feeding sessions were terminated at 300 sec. However, four of the seven animals tested failed to approach the bowl and were therefore assigned a maximum latency score of 300 sec. The remaining three animals consumed all pellets but at a significantly reduced rate compared to Day 1. Salivation, tremors, and fasciculations were observed in most animals at the time of testing. Twenty-four hours following the injection of sarin (Day 3), feeding rate and the total number of pellets consumed returned to predrug baseline levels but the average latency to begin feeding ( 11.4 set) remained significantly increased. In animals fed by hand, 12.5 &kg of sarin did not reduce total pellets consumed but increased the time taken to consume all pellets (Table 3). Salivation, tremors, fasciculations, piloerection, and diarrhea were observed in most animals at the time of testing. Activity Monitoring For the purpose of direct comparison with the reaching study, the activity levels of animals at only 45-60 min following administration of sarin are reported. During this period
TABLE 2 EFFECTS OF SARIN UPON VISUALLY
GUIDED
REACHING
PERFORMANCE
IN MARMOSETS
(N = 7 OR 8)
Responses (% total trials given)’ Dose sarin (a/kg id
% LD50”
0 5 7.5 10 12.5
0 22.2 33.3 44.4 55.5
% RBC AChE b inhibition 75 88 97 >99
Successes
Failures (with contact)
Failures (no contact)
No attempts
79 88.3 54.8’ 52.3* 26.8*
16.6 9.7 11 16.6 6.2*
3.8 0.9 5 5.8 9.2
0.8 0.9 29.3’ 24.8* 57.8+
’ Based upon mortality data at 48 hr in separate groups of animals. b Data from separate groups of animals (unpublished information, CDE). Abbreviations: im-intramuscular; AChE-red blood cell acetylcholinesterase. ‘Animals were tested 1 hr following administration of sarin. * Indicate significant difference from performance on the previous day (p < 0.05).
RBC
BEHAVIORAL TABLE 3
TOXICITY
OF SARIN IN MARMOSETS
s173
animals to respond (i.e., increased no-attempts) following sarin was suggested therefore EF’FEC~SOF SARIN (12.5 fig/kg im) UPON FEEDING MOTIVATION IN MARMOSETS (N = 8) to reflect a change in either feeding motivation or gross mobility, and thus the influence of Sarin Saline Sarin sarin upon these behaviors was investigated. (-24 hr) (+1 hr) (+24 hr) The results from both bowl- and hand-feeding experiments suggested that sarin at a dose Time to consume 1.9 one apple pellet (kO.1) (3; (+::i) of 12.5 pg/kg did not alter feeding motivation (mean + SE) (preferred food) in marmosets, as indicated by the number of pellets consumed. If animals Total pellets eaten 100 91.5 100 (70 total were able to approach food or if accessto food presented) was not dependent upon this ability, then feeding motivation was not altered. These data Note. Animals were trained to consume apple pellets presented in close proximity to their mouths (see text for did suggest that gross mobility in a substantial proportion of animals may be reduced sedetails of procedure). * Indicates significant difference from saline control verely. This was confirmed in a separate ex(p < 0.05). periment where substantial decreases in activity were observed 45-60 min following the administration of sarin at 12.5 &kg. However, a significant decrease in activity was observed no change in activity was observed during this following a dosage of 17.5 &kg sarin but not period following the administration of sarin following a dosage of 7.5 &kg sarin. Activity at 7.5 pug/kg, a dose shown to produce severe was also decreased (43% of control level on disruption of visuomotor coordination (Table Day 1) following 12.5 rg/kg sarin but this did 2). These data did not suggest a simple relanot reach statistical significance. tionship between visuomotor coordination and locomotor activity in marmosets. It was DISCUSSION concluded that the disruption of visuomotor coordination observed in marmosets following The 48-hr intramuscular LD50 for sarin in sarin administration did not reflect an indirect marmosets was estimated to be 22.3 (17.9effect of this agent upon either feeding moti23.6) &kg. This value suggests that the sen- vation or gross mobility. sitivity of the marmoset to satin is greater than Previous work in this laboratory has shown that of rodent species and rabbits but is very that decreasing the time available for retrieval similar to that of the rhesus monkey (unpubor eliminating one of the many anticipatory lished observations), which is regarded as a cues increases the number of no-attempts in good model for humans. drug-free animals (D’Mello et al., 1985). These Doses of sarin ranging from 33 to 55% LD50 data suggested that animals may not attempt were shown to disrupt visually guided reaching to retrieve if the task is “perceived” as being performance, a disruption characterized by “too difficult.” Furthermore, drugs may alter decreases in successful retrievals with con- the animals’ perception of the task and/or their comitant increases in no-attempts. However, own ability when drugged (unpublished obthis pattern of disruption did not suggest a servations). A similar suggestion has been specific effect of sarin upon visuomotor co- made in previous work to assessthe influence ordination. Such an action has been suggested of diazepam (Wetherell, 1979) and carbon for diazepam, chlorpromazine, and pentobarmonoxide (Mihevic et al., 1983) upon psybital, where decrements in performance were chomotor task performance in humans. The relevance of this hypothesis for the interpreobserved in the absence of increased no-attempts (D’Mello et al., 1985). The failure of tation of sarin-induced disruption of visuo-
s174
D’MELLO
motor coordination in marmosets is the subject of a current investigation in this laboratory. The return to control levels of performance observed 24 hr following administration of sarin in the various behavioral models studied provides further support for the view that signs of organophosphate poisoning during the recovery phase are not closely related to the degree of erythrocyte AChE inhibition. ACKNOWLEDGMENTS The authors thank Miss S. S. Miles, Mrs. T. M. Baker, and Mrs. E. J. Robinson for their excellent technical sup port throughout the course of these experiments.
REFERENCES BRACK, M., AND ROTHE, H. (1982). Organophosphate poisoning in marmosets. Lab. Anim. 16, 186-188. BRIMBLECOMBE, R. W. (1974). Drug Actions on Cholinergic Systems, pp. 64- 132. McMillan Press, New York. CAMPOS, F., ANDARRUDA, F. (198 1). Pharmacologic manipulations of brain catecholamines and the behaviour of Callithrix Jacchus (marmoset). Psychopharmacology 73,252-2x. D’MELLO, G. D., DUFFY, E. A. M., AND MILES, S. S. (1985). A conveyor belt task for assessing visuo-motor coordination in the marmoset (Callithrix jacchus): Effects of diazepam, chlorpromazine, pentobarbital and d-amphetamine. Psychopharmacology 86, 125- 13 1. D’MELLO, G. D., LEWANDOWSKA, E. A. M., ANDMILES, S. S. (1983). Effectsof the acetylcholinesterase inhibitor sarin upon a visually guided reaching response in the marmoset (Hapale Jacchus). In Application ofBehavioural Pharmacology in Toxicology (G. Zbinden, V. Cuomo, G. Racagni, and B. Weiss, eds.), pp. 153-156. Raven Press, New York. ELLMAN, G. L., COURTNEY, K. D., ANDRE& V., AND FEATHERSTONE, R. M. (1961). A new and rapid colorimetric determination of acetylcholinestemse activity. Biochem.
Pharmacol.
7,88-95.
FATEHYABALI, S., CHANDRA, O., ANDHASAN, M. (1980). Effectsof an organophosphate (dichlorvos) on open field behaviour and locomotor activity: Correlation with re-
AND DUFFY gional brain monoamine levels. Psychopharmacology 68,37-42. FINNEY, D. J. (197 1). Probit Analysis, 3rd ed. Cambridge Univ. Press, Cambridge. GROB, D. (1963). Anticholinesterase intoxication in man and its treatment. In Handbook of Experimental Pharmacology (G. B. Koelle, ed.), pp. 989-1027. SpringerVerlag, Berlin. HAMM, M. (1984). Chemical Warfare: The Growing Threat To Europe. Alliance, London. HOBBIGER,F. (1976). Pharmacology ofantichohnesterase drugs. In Handbook of Experimental Pharmacology (E. Zaimis, ed.), Vol. 42, pp. 487-581. Springer-Verlag, Berlin. LANDAUER, M. R., AND ROMANO, J. A. (1984). Acute behavioural toxicity of the organophosphate sarin in rats. Neurobehav. Toxicol. Teratol. 6,239-243. LAPORTE, R. E., KULLER, L. H., KUPFER, D. J., MCPARKLAND, R., MATTHEWS, G., AND CASPERSEN, C. ( 1979). An objective measure of physical activity for epidemiologic research. Amer. .I. Epidemiol. 109, 158168. MIHEVIC, P. M., GLINER, J. A., AND HORVATH, S. M. (1983). Carbon monoxide exposure and information processing during perceptual-motor performance. In?. Arch.
Occup. Environ.
Health
51, 355-363.
Ministry of Defence, U. K. (1972). Medical Manual of Dej&ce Against Chemical Agents, 5th ed. Her Majesty’s Stationery Office, London. SCRAGGS,P. R.. AND RIDLEY, R. M. (1978). Behavioural effectsof amphetamine in a small primate: Relative potencies of the d- and l-isomers. Psychopharmacology 59,243-245. SIEGEL, S. (1956). Non-Parametric Statistics for the Behavioural Sciences. McGraw-Hill, New York. TILSON, H. A., CABE, P. A., AND BURNE, T. A. (1980). Behavioural procedures for the assessment of neurotoxicity. In Experimental and Clinical Neurotoxicology (P. S. Spencer and H. H. Schaumberg, eds.), pp. 758766. Williams and Wilkins, New York. WEISS, B., AND LATIES, V. G. (1975). Behavioural Toxicology. Plenum, New York. WETHERELL, A. (1979). Individual and group effects of 10 mg diazepam on drivers’ ability, confidence and willingness to act in a gapjudging task. Psychopharmacology
63,259-267.
WINER, B. J. ( 197 1). Statistical Principles in Experimental Design, 2nd ed. McGraw-Hill, London. WOLTHIUS, 0. L., AND VANWERSCH,R. A. P. (1984). Behavioural changes in the rat after low doses of cholinesterase inhibitors. Fundam. Appl. Toxicol. 4, S195S208.
AND
APPLIED
TOXICOLOGY
5,
S 169-S
174 (
1985)
The Acute Toxicity of Sarin in Marmosets (Callithrix jacchus): A Behavioral Analysis G. D. D'MELLO' AND E. A. M. DUFFY Chemical Defence Establishment, Porton Down, Salkbury, Wiltshire, United Kingdom SP4 OJQ
The Acute Toxicity of Satin in Marmosets (Callithrir jacchus): A Behavioral Analysis. D’MELLO, G. D., AND DUFFY, E. A. M. (1985). Fundam. Appl Toxicol. 5, Sl69-S 174. In marmosets, the clincial signs of poisoning following the administration of satin and the effects of satin upon performance in three behavioral test models were investigated. The sensitivity of marmosets to the lethal action of satin was shown to be greater than that of rodent species and rabbits but very similar to that of the rhesus monkey. Doses of sarin ranging from 33 to 55% LD50, resulting in erythrocyte acetylcholineatemse inhibitions of 88% or mom, were shown to disrupt the performance of a food-reinforced visually guided reaching response. However, the pattern of disruption did not suggest a specific action of satin upon visuomotor coordination and could not be explained on the basis of an indirect effect of this agent upon either feeding motivation or changes in gross mobility. It was suggested that possible changes in animals’ perception of the reaching task and/ or their own drugged ability may be relevant to any interpretation of satin-induced changes in visuomotor coordination in this species. Q 1985 Academic Pres, Inc.
Sublethal doses of organophosphorus compounds are known to produce symptoms likely to degrade normal function in humans (Brimblecombe, 1974). Research methods are therefore required to characterize those sublethal, and in particular the behavioral, effects of those organophosphorus compounds which are possible chemical warfare agents (Hamm, 1984). One traditional approach involves the selection of a rodent species such as the rat and a wide range of neurobehavioral test models (Tilson et al., 1980; Weiss and Laties, 1975). However, the relevance of rodent behavioral models for humans is debatable. Although the so-called higher primates are regarded generally as the species of choice, they are not readily available to the majority of experimenters. A requirement therefore exists for the development of neurobehavioral test methods in a primate species that is readily available.
’ To whom correspondence should be addressed.
The work to be described here represents part of a research program to characterize the behavioral effects of sarin in marmosets (Cullithrix jacchus), a primitive primate but one which is gaining popularity in behavioral pharmacological research (Campos and Arruda, 1981; D’Mello et al., 1985; Scraggs and Ridley, 1978). To facilitate interspecies comparison the lethal potency of sarin and the incidence and duration of signs of poisoning were determined. On the basis of symptoms observed in humans following mild poisoning by acetylcholine&erase (AChE) inhibitors (i.e., <50% erythrocyte AChE inhibition), (Ministry of Defence, 1972) and consideration of the effects of these agents upon neuromuscular function (Hobbiger, 1976), changes in voluntary motor output are expected. AChE inhibitors have been reported to disturb a variety of motor behaviors in both rodent (Fatehyab et al., 1980; Landauer and Romano, 1984; Wolthuis and Vanwersch, 1984) and primate (Brack and Rothe, 1982; Grob, 1963) species.
S169
0272-0590/85 $3.00 Copyright 8 1985 by the Sociely of Toxicology. All rights of reproduction in any form d.
s170
D’MELLO
However, the effects of these agents upon fine motor control have not been investigated fully. The influence of sarin upon a food-reinforced, visually guided reaching response has therefore been investigated using a method developed previously in this laboratory (D’Mello et al., 1985). Since performance on this task may be influenced by a number of behavioral components, notably feeding motivation and gross mobility, ancillary experiments were conducted to assessthese factors and also to provide a better understanding of those behavioral components mediating op timal performance on this task. A preliminary account of some of these data has been reported (D’Mello et al., 1983). METHODS Animnls. Marmosets of both sexesbred at the Chemical Defence Establishment were used. Each animal was housed individually in a cage (750 X 480 X 600 mm) located within a room maintained at 22-23°C and at a relative humidity of SO-60%, with a regular day-night cycle imposed by electric lighting (light from 7%) AM to 7:OOPM). All subjects were maintained at their free feeding body weights (range 290-380 g) on a high-protein diet (Mazuri primate diet, SDS, Witham, Essex, U.K.) with fruit and vitamin supplement. Apple and banana were withheld from those animals used in either the visually guided reaching or feeding motivation studies. Acute toxicity. Each animal was administered one of five doses of satin (range 15-25 &kg) and the signs of poisoning were monitored as a function of time up to 48 hr. No attempt was made to either quantify the intensity of the signs observed or to distinguish between various sign subtypes (e.g., clonic or tonic convulsions, resting or intentional tremors). At 48 hr, the number of deaths was recorded. Visually guided reaching. Marmosets were trained to retrieve small cylinders of apple from a moving belt positioned in front of their home cages. Access to the belt was restricted and belt velocity increased progressively during training to impose a maximum response time of less than 0.5 sec. Belt direction was changed on alternate trials, of which there were 50 each session. Responses were categorized according to four criteria. A “successful retrieval” was recorded if the animal caught and then ingested the pellet. A “failure” with or without contact was recorded when animals reached for but failed to retrieve the pellet. A “no-attempt” was recorded when animals did not respond. A detailed description of the apparatus and procedure has been reported (D’Mello et aZ., 1985).
AND DUFFY On the basis of data collected during the final week of training, five groups (n = 7 or 8) were selected, matched for performance, and allocated randomly to one of five treatments (saline or sarin 57.5, 10, or 12.5 pg&g). Testing was conducted on three consecutive days. On Day 1, all groups received saline. On Day 2, each group received their allocated treatment. On Day 3, all groups were tested for any residual treatment effects. Feeding motivation. Marmosets were trained to consume a fixed number (49 or 50) of apple pellets within a 5-min period. One group (n = 7) was presented with pellets in a feeding bowl placed within their home cages.Latency to begin feeding, the cumulative duration of feeding, and the number of pellets consumed were recorded. In a second group (n = 8) each animal was removed from its home cage and held within the gloved hands of an experimenter such that voluntary movements of the upper trunk, arms, and head were relatively unrestricted. A second experimenter presented pellets singly to the animal via a purposebuilt dispenser. Pellets were presented in close proximity to the animal’s mouth (2-3 cm) at a rate determined by the speed at which each animal was able to consume the pellets. Animals received a single feeding session each day until stable baseline levels of feeding were established. Testing was conducted on three consecutive days as described for those groups trained on the reaching task. The influence of a single dose level of satin (12.5 pg/kg) was assessed and corresponded to the highest dose of satin tested in the reaching task. The total number of pellets available and session time were selected to facilitate direct comparison with the reaching experiment. It was hypothesized that any changes in feeding motivation, as indicated by the number of pellets consumed, mediated indirectly by changes in gross mobility, would manifest in the “bowlfeeding” group but not in the “hand-feeding” group. Activity monitoring. The gross mobility or locomotor activity of groups of marmosets (n = 4) was monitored using a device developed previously to monitor activity levels in humans (Laporte et al. 1979). The device (37 X 30 X 13 mm; weight I9 g) was mounted on the back of an animal by means of a simple elastic harness. Movements by the animal in the anterorostral plane operated a simple mercury tilt switch, closures of which were used as activity counts. Counts were cumulated and stored by integrated circuitry and could be displayed in digital form following the manual operation of a separate magnetic switch. To monitor changes in the level of activity as a function oftime, the animal was caught by an experimenter at various times over a period of 4 hr and cumulated counts recorded. Testing was conducted within the home cage of each animal over three consecutive days. On Day 1, the device was fitted and each animal given 24 hr to habituate to its presence. On Day 2, control readings were taken. On Day 3, animals were injected with either saline or satin (7.5, 12.5, or 17.5 rcglkg), and readings were taken at times corresponding to those of Day 2.
BEHAVIORAL
TOXICITY
s171
OF SARIN IN MARMOSETS
Drug administration. Satin was synthesized by the Organic Chemistry Section at Chemical Defence Establishment. Dilutions were made with 0.9% saline solution. All injections were given intramuscularly in the inner thigh in a volume of 0.5 ml/kg body wt. In the visually guided reaching and feeding motivation tests, saline or sarin was administered 60 min before test sessions. This test time was selected to correspond with the time of peak erythrocyte AChE inhibition determined as described below. AChE activity. Blood samples were taken from the tail vein of separate groups of animals and then assayed for erythrocyte AChE activity using the spectrophotometric method of Ellman et al. (1961). Acetylthiocholine (1.0 mM) was used as the substrate. Statistical analysis. Statistical methods to determine group differences and appropriate to the nature of the data analyzed were selected according to the criteria described by Siegel (1956). Interval data (i.e., temporal scores) were first subjected to homogeneity of variance tests (F tests) and, if homogenous, compared using either paired or unpaired t tests asappropriate (Winer, 1971). For nonhomogenous data, groups were compared using either the Wilcoxon matched pairs, signed ranks test or the KolmogorovSmirnov two-sample test as appropriate (Siegel, 1956). Categorical data (i.e., frequency scores) were analyzed by means of x2 or Fisher’s exact probability tests(Siegel, 1956). LD50 values, based upon mortality data at 48 hr, were estimated using probit analysis (Finney, 197 1).
tion, and unsteadiness were observed in the majority of animals irrespective of dose. The first four of these signs were continuous in nature and were generally observed for periods in excess of 4 hr following poisoning. Bouts of increased mastication, swallowing, and nictitation were also observed frequently. There was a low incidence of vomiting (11%) and diarrhea (22% of animals tested). There were no overall differences in the frequency of signs observed between animals that received high (25 &kg) or low (15-20 pg/kg) doses of sarin. These doses corresponded to the estimated LD 100 and LD 10 doses for sarin, respectively. The incidence of vomiting and diarrhea seemed higher in those animals that received low doses, but this did not reach statistical significance. The first signs observed ca. 20 set following intramuscular injection of sarin were generally tremors, unsteadiness, or increased mastication. The times to onset of the first sign and of tremors and convulsions were significantly lower in animals that received the high dose of sarin (Table 1).
RESULTS Acute Toxicity
Visually Guided Reaching
The 48-hr intramuscular LD50 for sarin in marmosets was estimated to be 22.3 (17.923.6) &kg. Salivation, tremors, fasciculations, loss of posture, convulsions, piloerec-
Baseline levels of performance on this task for each of the five groups tested following saline administered on Day 1 did not differ. About 85% of total trials resulted in successful
TABLE 1 MEAN
TIME (MIN)
Dose of satin k&g)
TO ONSET OF FIRST OBSERVED AND MOST FREQUENT SIGNS FOLLOWING ADMINISTRATION OF SARIN IN MARMOSETS (N = 6 OR 9)
First observed sign
Salivation
15-20 (
1.9 (kO.6)
(,E)
(L&O)
(228;
(3)
Tremor
INTRAMUSCULAR
Convulsions 8.6 (e1.7)
,,i::;
(2::;
Note. Two animals in the 25 &kg dose group did not convulse. Numbers in parentheses show + 1 SE. Asterisks indicate significant difference between dose levels (p < 0.05).
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D’MELLO
AND DUFFY
retrievals. Animals responded but failed to retrieve on about 12% of trials. One hour following the injection of sarin, at doses resulting in erythrocyte AChE inhibitions of 88% or more, the number of successful retrievals was decreased in a dose-related manner (Table 2). At doses of satin between 7.5 and 12.5 &kg decreases in successes were accompanied by increases in no-attempts with little evidence for an increase in failures. Although signs of poisoning were not monitored routinely during this study, tremors and fasciculations were observed frequently both before and during testing. Twenty-four hours following the injection of sarin (Day 3), performance on the reaching task returned to predrug baseline levels in all groups (not shown). Feeding Motivation One hour following saline administration (Day 1) animals presented with 50 apple pellets in a bowl placed in their home cages began feeding within an average 2.4 set and consumed all pellets at an average rate of 20/min. One hour following administration of 12.5 & kg of satin on Day 2, these same animals took an average 208 set to begin feeding and con-
sumed a median of 20 pellets before feeding sessions were terminated at 300 sec. However, four of the seven animals tested failed to approach the bowl and were therefore assigned a maximum latency score of 300 sec. The remaining three animals consumed all pellets but at a significantly reduced rate compared to Day 1. Salivation, tremors, and fasciculations were observed in most animals at the time of testing. Twenty-four hours following the injection of sarin (Day 3), feeding rate and the total number of pellets consumed returned to predrug baseline levels but the average latency to begin feeding ( 11.4 set) remained significantly increased. In animals fed by hand, 12.5 &kg of sarin did not reduce total pellets consumed but increased the time taken to consume all pellets (Table 3). Salivation, tremors, fasciculations, piloerection, and diarrhea were observed in most animals at the time of testing. Activity Monitoring For the purpose of direct comparison with the reaching study, the activity levels of animals at only 45-60 min following administration of sarin are reported. During this period
TABLE 2 EFFECTS OF SARIN UPON VISUALLY
GUIDED
REACHING
PERFORMANCE
IN MARMOSETS
(N = 7 OR 8)
Responses (% total trials given)’ Dose sarin (a/kg id
% LD50”
0 5 7.5 10 12.5
0 22.2 33.3 44.4 55.5
% RBC AChE b inhibition 75 88 97 >99
Successes
Failures (with contact)
Failures (no contact)
No attempts
79 88.3 54.8’ 52.3* 26.8*
16.6 9.7 11 16.6 6.2*
3.8 0.9 5 5.8 9.2
0.8 0.9 29.3’ 24.8* 57.8+
’ Based upon mortality data at 48 hr in separate groups of animals. b Data from separate groups of animals (unpublished information, CDE). Abbreviations: im-intramuscular; AChE-red blood cell acetylcholinesterase. ‘Animals were tested 1 hr following administration of sarin. * Indicate significant difference from performance on the previous day (p < 0.05).
RBC
BEHAVIORAL TABLE 3
TOXICITY
OF SARIN IN MARMOSETS
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animals to respond (i.e., increased no-attempts) following sarin was suggested therefore EF’FEC~SOF SARIN (12.5 fig/kg im) UPON FEEDING MOTIVATION IN MARMOSETS (N = 8) to reflect a change in either feeding motivation or gross mobility, and thus the influence of Sarin Saline Sarin sarin upon these behaviors was investigated. (-24 hr) (+1 hr) (+24 hr) The results from both bowl- and hand-feeding experiments suggested that sarin at a dose Time to consume 1.9 one apple pellet (kO.1) (3; (+::i) of 12.5 pg/kg did not alter feeding motivation (mean + SE) (preferred food) in marmosets, as indicated by the number of pellets consumed. If animals Total pellets eaten 100 91.5 100 (70 total were able to approach food or if accessto food presented) was not dependent upon this ability, then feeding motivation was not altered. These data Note. Animals were trained to consume apple pellets presented in close proximity to their mouths (see text for did suggest that gross mobility in a substantial proportion of animals may be reduced sedetails of procedure). * Indicates significant difference from saline control verely. This was confirmed in a separate ex(p < 0.05). periment where substantial decreases in activity were observed 45-60 min following the administration of sarin at 12.5 &kg. However, a significant decrease in activity was observed no change in activity was observed during this following a dosage of 17.5 &kg sarin but not period following the administration of sarin following a dosage of 7.5 &kg sarin. Activity at 7.5 pug/kg, a dose shown to produce severe was also decreased (43% of control level on disruption of visuomotor coordination (Table Day 1) following 12.5 rg/kg sarin but this did 2). These data did not suggest a simple relanot reach statistical significance. tionship between visuomotor coordination and locomotor activity in marmosets. It was DISCUSSION concluded that the disruption of visuomotor coordination observed in marmosets following The 48-hr intramuscular LD50 for sarin in sarin administration did not reflect an indirect marmosets was estimated to be 22.3 (17.9effect of this agent upon either feeding moti23.6) &kg. This value suggests that the sen- vation or gross mobility. sitivity of the marmoset to satin is greater than Previous work in this laboratory has shown that of rodent species and rabbits but is very that decreasing the time available for retrieval similar to that of the rhesus monkey (unpubor eliminating one of the many anticipatory lished observations), which is regarded as a cues increases the number of no-attempts in good model for humans. drug-free animals (D’Mello et al., 1985). These Doses of sarin ranging from 33 to 55% LD50 data suggested that animals may not attempt were shown to disrupt visually guided reaching to retrieve if the task is “perceived” as being performance, a disruption characterized by “too difficult.” Furthermore, drugs may alter decreases in successful retrievals with con- the animals’ perception of the task and/or their comitant increases in no-attempts. However, own ability when drugged (unpublished obthis pattern of disruption did not suggest a servations). A similar suggestion has been specific effect of sarin upon visuomotor co- made in previous work to assessthe influence ordination. Such an action has been suggested of diazepam (Wetherell, 1979) and carbon for diazepam, chlorpromazine, and pentobarmonoxide (Mihevic et al., 1983) upon psybital, where decrements in performance were chomotor task performance in humans. The relevance of this hypothesis for the interpreobserved in the absence of increased no-attempts (D’Mello et al., 1985). The failure of tation of sarin-induced disruption of visuo-
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D’MELLO
motor coordination in marmosets is the subject of a current investigation in this laboratory. The return to control levels of performance observed 24 hr following administration of sarin in the various behavioral models studied provides further support for the view that signs of organophosphate poisoning during the recovery phase are not closely related to the degree of erythrocyte AChE inhibition. ACKNOWLEDGMENTS The authors thank Miss S. S. Miles, Mrs. T. M. Baker, and Mrs. E. J. Robinson for their excellent technical sup port throughout the course of these experiments.
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