VETERINARSKI ARHIV 69 (1), 29-37, 1999

ISSN 1331-8055 Online
ISSN 0372-5480 Printed in Croatia

Reduction of dichlorvos-induced toxicosis in
rabbits by levamisole

Imad Ibrahim Aldabagh and Fouad Kasim Mohammad*

Department of Physiology, Biochemistry and Pharmacology,
College of Veterinary Medicine, University of Mosul, Iraq

* Contact address:
Prof. Dr. Fouad Kasim Mohammad,
Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Mosul, P.O. Box 11136, Mosul, Iraq

ALDABAGH, I. I., F. K. MOHAMMAD: Reduction of dichlorvos-induced toxicosis in rabbits by levamisole. Vet. arhiv 69, 29-37, 1999.

ABSTRACT

The toxicity and interaction of levamisole with dichlorvos were examined in male rabbits. Signs of levamisole (25, 50 and 75 mg/kg, subcutaneously) poisoning in rabbits were dose-dependent, and the highest dose produced excessive salivation, lacrimation, ataxia and tremors in all the rabbits. This dose induced convulsion and death in 50 and 75% of the rabbits, respectively. Levamisole at 25 and 75% did not significantly affect erythrocyte acetylcholinesterase (EChE) activity at 2 and 24 h after treatment. Dichlorvos dosing at 10 and 20 mg/kg, orally dose-dependently induced signs of cholinergic toxicity. The 20 mg/kg dose of dichlorvos caused death in 75% of the rabbits. Levamisole pretreatment at 50 mg/kg protected the rabbits against dichlorvos (20 mg/kg, orally) induced toxicosis, prevented the occurrence of convulsion, gasping and death, and significantly increased the latency to onset of tremor in comparison with the dichlorvos-treated control group. Combined levamisole (50 mg/kg) and dichlorvos (10 mg/kg) treatments caused a significantly higher degree of EChE inhibition at 2 and 24 h post-treatment in comparison with the dichlorvos-treated group. The data suggest that levamisole protects rabbits intoxicated with dichlorvos, irrespective of the extent of EChE inhibition.

Key words: levamisole, dichlorvos, rabbit, erythrocyte cholinesterase symptoms, organophosphate

Introduction

Levamisole, the 1-isomer of tetramisole, is an anthelmintic used in veterinary practice (MARRINER and ARMOUR, 1986; DALVI, 1990). Levamisole poisoning in animals is characterised by salivation, lacrimation, miosis, hyperventilation or dyspnea, frequent urination and defecation, ataxia, tremors and convulsion before death (HSU, 1980, 1981a, 1981b; DALVI, 1990). These signs of levamisole poisoning resemble those of organophosphorus (OP) insecticides which cause muscarinic, nicotinic and central nervous system effects (ANON., 1986; FIKES, 1990). Thus, it appears that there is the possibility of potentiated toxic effects when levamisol and OP insecticides are concurrently used in animals.

Treatment of mice (HSU, 1980) and pig (HSU, 1981a) with the PO insecticide dichlorvos did not increase the toxicity of levamisole. Similarly, the PO insecticide diazinon did not increase the toxicity of tetramisole in sheep (FORSYTH, 1966). However, in these studies the insecticides were given prior to levamisole (HSU, 1980, 1981a) or tetramisole (FORSYTH, 1966). The present study was undertaken in rabbits in order to further examine levamisole-induced toxicosis and its interaction with dichlorvos, taking account of the administration of levamisole before dichlorvos dosing. This approach provided some new information about the ability of levamisole in reducing the toxic effects of dichlorvos.

Materials and methods

Male cross-bred rabbits with masses of 1.2-1.9 kg were used. They were housed at a temperature of 22±2 °C with 10/14 h light/dark cycle. The animals had free access to food and water.

Experiment I, toxic effects of levamisole:

Rabbits (n=4/group) were treated subcutaneously (s.c.) with levamisole hydrochloride (7.5%, Phenex, Belgium) at dose rates of 25, 50 and 75 mg/kg body mass. The control group received physiological saline solution at 1 ml/kg, s.c. Levamisole doses were determined in a preliminary experiment in rabbits. After levamisole injection, each rabbit was observed until death, or for 2 h, for the appearance of signs of poisoning which included salivation, lacrimation, frequent urination and defecation, ataxia, tremor and convulsion.

Two and 24 h after levamisole administration (25 and 50 mg/kg, s.c.) blood samples were obtained from the ear vein by a heparinised syringe. Erythrocytes were separated by centrifugation of the blood samples at 3000 rpm for 15 minutes. An aliquot of 0.1 ml of erythrocytes was used for the determination of erythrocyte acetylcholinesterase (EChE) activity by the modified electrometric method described previously (MOHAMMAD and OMER, 1982).

Experiment II, effects of levamisole pre-treatment on dichlorvos-induced toxicosis:

In this experiment, 4 rabbits/group were used. Levamisole was administered at dose rates of 0 (saline-control), 25 and 50 mg/kg, s.c. 30 minutes before dosing of the rabbits with dichlorvos (Aforfos 50% EC, K.N. Efthymiadis, Greece) at 10 mg/kg, orally. Also, rabbits were treated with levamisole at 0 (saline-control) and 50 mg/kg, s.c. 30 minutes before dosing with a higher dose of dichlorvos (20 mg/kg, orally). The doses of dichlorvos were predetermined to produce cholinergic toxicity in rabbits. Dichlorvos was diluted with distilled water and administered by a gavage needle to a volume of 0.5 ml/kg body mass. Following dichlorvos dosing, each rabbit was observed until death, or for 2 h, for the occurrence of signs of toxicosis characteristic of cholinergic hyperstimulation (ANON., 1986; FIKES, 1990; FARIS and MOHHAMAD, 1997). The severity score of toxicity in rabbits doses with dichlorvos was determined by adding the assigned grades to the signs of toxicosis: 0=normal, 1=hyperactivity and/or agitation, 2=salivation, 3=tremor, 4=muscle fasciculation, 5=flat body appearance, 6=convulsion, 7=death.

The latencies to onset of tremor and death within 2 h were recorded. The activity of EChE was determined as mentioned in experiment I prior to drug administration (time 0, baseline) and thereafter 2 and 24 h after dichlorvos dosing at 10 mg/kg. The percent of EChE inhibition was determined as follows:

% inhibition= SEChE activity (baseline)-EChE activity at 2 or 24 h/EChE activity (baseline)C × 100

In both experiments, the rabbits were retained for 3 days for any delayed death.

Statistics: Frequency data were subjected to Fisher's exact probability test (RUNYON, 1977). The severity score to toxicity was analysed by Mann-Whitney-U-test (RUNYON, 1977). Tremor times were compared by Unpaired Student's-t-test (BRUNING and KINTZ, 1977). The level of significance was at P<0.05.

Results

Subcutaneous injection of levamisole in rabbits induced signs of poisoning that were dose dependent (Table 1). At the highest dose (75 mg/kg) tested, levamisole significantly increased the occurrence of excessive salivation, lacrimation, ataxia and tremor by 100%, and caused convulsion and death in 50 and 75% of the rabbits, respectively (Table 1). The mean±SE latency to onset of death was 55±20 minutes. The 50 mg/kg dose of levamisole was well tolerated by the rabbits which showed salivation (75%), lacrimation (50%) as well as ataxia and tremor (25%) in comparison with the control group (Table 1). Levamisole at 25 and 50 mg/kg did not significantly affect EChE activity at 2 and 24 h after treatment (Table 2). However, in the levamisole-treated rabbits EChE activity was decreased by 16-18%, at 2 h post-treatment, in comparison with the control value (Table 2).

Table 1. Levamisole-induced toxicosis in rabbits

N=4 rabbits/dose group. Each rabbit was observed until death or for 2 h after levamisole treatment.
*Significantly different from the corresponding control value, P<0.05.

Table 2. Erythrocyte acetylcholinesterase activity in rabbits treated with levamisole

Dichlorvos dosing at 10 or 20 mg/kg in rabbits dose-dependently induced signs of toxicosis characteristics of cholinesterase inhibition and subsequent cholinergic overstimulation (Table 3). These signs were salivation, lacrimation, frequent urination and defecation, tremor, ataxia, muscle fasciculation, flat body appearance, gasping and convulsion (Table 3). The 20 mg/kg dose of dichlorvos caused death in 3 out of 4 rabbits. The latency to onset of death was 25±7 minutes.

Table 3. Effect of levamisole on dichlorvos-induced toxicosis in rabbits

Rabbits (4/group) were pretreated with levamisole 30 minutes before dichlorvos dosing. Each rabbit was observed until death or for 2 h after dichlorvos dosing. *The severity score of toxicity for each rabbit was the summation of grades of sign of toxicosis from 0 to 7: 0=normal; 1=hyperactivity and/or agitation; 2=salivation; 3=tremor; 4=muscle fasciculation; 5=flat body appearance; 6=convulsion; 7=death. For reference, the highest severity score when all the signs of toxicosis mentioned above are observed in an individual rabbit would be 28. *Significantly different from the respective control (0 dose) value, P<0.05.

Levamisole pre-treatment protected the rabbits against dichlorvos (10 or 20 mg/kg) induced toxicosis, and the median score of the severity of toxicosis significantly decreased in comparison with the respective control non-levamisole-treated groups (Table 3). The protective effect of levamisole was best seen at the dose rate of 50 mg/kg when given before the high dose (20 mg/kg) of dichlorvos. At these doses of both drugs, levamisole decreased the occurrence of frequent urination, muscle fasciculation and flat body appearance, but most importantly, levamisole blocked the occurrence of convulsion, gasping and death induced by dichlorvos (Table 3). Levamisole at 50 mg/kg significantly prolonged the latency to onset of tremor in the dichlorvos-treated group when compared with saline-dichlorvos (10 or 20 mg/kg) treated groups (Table 4).

Table 4. Effect of levamisole on the latency to onset of tremors in rabbits dosed with dichlorvos

The values are mean±SE of 4 rabbits/group. Levamisole was injected subcutaneously 30 minutes before oral dosing of dichlorvos. *Significantly different from the respective control value, P<0.05.

Fig. 1. Percentage of erythrocyte acetylcholinesterase (EChE) inhibition in rabbits dosed orally with dichlorvos (10 mg/kg) after pre-treatment with saline (control) and levamisole at 25 and 50 mg/kg, s.c.. Pre-treatment (baseline) values (mean±SE) of EChE activity of the three groups were 0.44±0.02, 0.38±0.03 and 0.41±0.02, difference pH/30 minutes, respectively. %inhibition=SEChE activity (baseline)-EChE activity at 2 or 24 h / EChE activity (baseline)C × 100. Levamisole was injected 30 minutes before dichlorvos dosing. *Significantly different from the control (dichlorvos-saline) group, P<0.05.

Combined levamisole (50 mg/kg) and dichlorvos (10 mg/kg) treatments caused a significantly higher degree of EChE inhibition at 2 and 24 h after dichlorvos dosing in comparison with the dichlorvos-treated group (Figure 1).

Discussion

Levamisole produced signs of toxicosis in rabbits characteristic of cholinergic overstimulation. These effects were in agreement with those reported in sheep treated with tetramisole (FORSYTH, 1966; PHILIP and STONE, 1967) or pigs and mice treated with levamisole (HSU, 1980; 1981a). The findings of the present study do not support the hypothesis that levamisole, owing to its cholinomimetic effects, could potentiate the toxicity of organophosphates. As found in Table 3, levamisole prevented the occurrence of convulsion, gasping and death in rabbits dosed with dichlorvos at 20 mg/kg. The results further support the findings of others, who found that there was no adverse interaction between dichlorvos and levamisole (HSU, 1980; 1981a). However, our results extend these findings and further suggest that levamisole possesses a protective effect against dichlorvos-induced toxicosis and lethality in rabbits. The clinical implication of this study is that levamisole could be safely used with OP insecticides in animals that need combined anthelmintic and insecticidal therapy. Further field studies, however, are needed in target animal species, such as ruminants, to verify and support this point.

The mechanism of the protective effect of levamisole against dichlorvos-induced toxicosis is not clear at present. Tetramisole (EYRE, 1970) and levamisole (ALDABAGH and MOHAMMAD, 1998) were found to inhibit acetylcholinesterase activity in vitro. Considering the fact that levamisole at 25 and 50 mg/kg decreased (though non-significantly) EChE in the rabbits by 18 and 16%, respectively (Table 2), it is therefore possible that levamisole could reversibly inhibit acetylcholinesterase in vivo and prevent or delay the irreversible inhibition of this enzyme by dichlorvos. Such a protective mechanism is found between physostigmine and organophosphates (SOMANI and DUBE, 1989). One difficulty in accepting this hypothesis is that levamisole did not significantly inhibit EChE in the rabbits of the present study or in pigs (HSU, 1981a). But, levamisole pre-treatment significantly increased the inhibitory effect of dichlorvos on EChE, suggesting that levamisole and dichlorvos may bind at different sites of the enzyme. Furthermore, the calculated percentages of EChE inhibition suggest that levamisole pre-treatment did not potentiate the anticholinasterase action of dichlorvos in rabbits. Also, in vitro inhibition of rabbit plasma and erythrocyte cholinesterase by a combination of levamisole and dichlorvos did not exceed the sum of inhibitions induced by either drug alone (ALDABAGH and MOHAMMAD, 1998). The findings of the present study with regard to cholinesterase inhibition correlate with those reported in pigs treated with dichlorvos and levamisole (HSU, 1981a).

In conclusion, adverse drug interaction is not likely to occur in animals concurrently treated with levamisole and dichlorvos; instead, levamisole pre-treatment may diminish the toxic effects of dichlorvos.

Acknowledgement
This study was supported by the College of Veterinary Medicine, University of Mosul.

References

ALDABAGH, I. I., F. K. MOHAMMAD (1998): In vitro inhibition of rabbit blood cholinesterase by levamisole and dichlorvos. Iraqi J. Vet. Sci. 11, 17-22.

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Received: 13 August 1998
Accepted: 15 January 1999

ALDABAGH, I. I., F. K. MOHAMMAD: Smanjivanje toksicnosti diklorvosa u kunica levamisolom. Vet. arhiv 69, 29-37, 1999.

SAZETAK

Istrazivana je toksicnost diklorfosa i interakcije s levamisolom na muskim kunicima. Znakovi otrovanja diklorvosom (25, 50 i 75 mg/kg, potkozno) u kunica ovisili su o dozi, a najvisa doza je u svih uzrokovale pojacano slinjenje, suzenje, ataksiju i drhtanje. Ta doza uzrokovala je i konvulzije u 50% i smrt u 75% kunica. Levamisol na 25% i 75% nije znacajno utjecao na aktivnost acetilkolinesteraze eritrocita (EChE) 2 i 24 sata nakon trovanja. Oralne doze diklorvosa od 10 i 20 mg/kg izazvale su, ovisno o dozi, znakove kolinergicne toksicnosti. Doza od 20 mg/kg diklorvosa izazvala je smrt 75% kunica. Prethodno davanje levamisola u dozi od 50 mg/kg zastitilo je kunice od otrovanja diklorvosom (20 mg/kg), odnosno sprjecilo je pojavu konvulzija, dahtanja i smrti, a znacajno je povecalo latenciju pocetka drhtanja u usporedbi s kontrolnom skupinom tretiranom diklorvosom. Kombinirani tretman levamisolom (50 mg/kg) i diklorvosom (10 mg/kg) izazvao je znacajno visi stupanj EChE inhibicije na 2 i 24 sata nakon davanja u usporedbi sa skupinom koja je dobila samo diklorvos. Podaci upucuju na zakljucak da levamisol stiti kunice otrovane diklorvosom, neovisno o inhibiciji EChE.

Kljucne rijeci: levamisol, diklorvos, kunic, simptomi kolinesteraze eritrocita, organofosfati