Digitoxin

Abstract: The acute toxicity of dried Nerium oleander leaves to Najdi sheep is described in 12 sheep assigned as untreated controls, N. oleander-treated once at 1 and 0.25 g/kg body weight and N. oleander-treated daily at 0.06 g/kg body weight by drench. Single oral doses of 1 or 0.25 g of dried N. oleander leaves/kg body weight caused restlessness, chewing movements of the jaws, dyspnea, ruminal bloat, incoordination of movements, limb paresis, recumbency and death 4-24 hr after dosing. Lesions were widespread congestion or hemorrhage, pulmonary cyanosis and emphysema, hepatorenal fatty change and catarrhal abomasitis and enteritis. The daily oral doses of 0.06 g dried N. oleander leaves/kg body weight caused less severe signs and death occurred between days 3 and 14. In these animals, the main lesions were hepatonephropathy and gelatinization of the renal pelvis and mesentry and were accompanied by significant increases in serum AST and LDH activities, in bilirubin, cholesterol and urea concentrations and significant decreases in total protein and albumin levels, anemia and leucopenia.

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Nerium oleander, a member of the family Apocynaceae is a flowering plant that grows in Asia, Mediterranean regions and many tropical and subtropical countries (Watt and Breyer-Brandwijk, 1962; Migahid, 1978). The plant is locally known as Deflah and used in traditional medicine as a diuretic, cardiac tonic and for the treatment of hemorrhoids, eczema, epilepsy, snake bite and gastrointestinal disturbances (Ageel et al., 1987). Aerial parts of the plant contain cardiac glycosides, oleandroside, nerioside and digitoxin-type glycoside that are structurally similar to digitalis and known to cross react in digoxin radioimmunoassays (Osterloh et al., 1982). Haynes et al. (1985) reported death of a woman after drinking herbal tea prepared from N. oleander leaves and found that this case demonstrated the cross-reactivity between the cardiac glycosides in the plant and the digoxin radioimmunoassay.

Nerium oleander poisoning has been reported in cattle (Mahin et al., 1984), man and experimental canine models (Clark et al., 1991). Because N. oleander toxicosis has been suspected in grazing ruminants by livestock owners and because of the therapeutic utility of the plant against various ailments as well as the lack of appropriate information of the pathogenesis of N. oleander poisoning we investigated the effect of various oral doses of the plant leaves on Najdi sheep.

Methods

Animals and Plant Administration

Twelve 12-15 month-old clinically healthy male Najdi sheep were housed within the premises of the experimental farm station of the College of Agriculture and Veterinary Medicine, King Saud University at Buraidah and fed dried lucerne and barley grains and water was provided ad libitum. The sheep were assigned to 4 groups each of 3 sheep.

Sheep 1, 2 and 3 were the untreated controls (group 1). Nerium oleander leaves were collected from the nearby areas of Buraidah, shade-dried, ground with a mortar and pestle and given in single oral doses of 1 g/kg body weight to sheep 4, 5 and 6 (group 2) and 0.25 g/kg body weight to sheep 7, 8 and 9 (group 3) and in repeated daily oral doses of 0.06 g/kg body weight to sheep 10, 11 and 12 (group 4).

Clinical signs and mortality rates were recorded. Necropsy findings were described immediately after death and specimens of CNS, peripheral nerves, lungs, trachea, heart, liver, gall bladder, kidneys, urinary bladder, spleen, abomasum and intestines were fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned at 6 [micro]m and stained with hematoxylin and eosin (H&E) for histopathological examination. The gross lesions were scored according to their severity on a-to +++ scale as described earlier (Abdel Gadir and Adam, 1999) and compared to microscopic changes.

Laboratory Assays

The sheep were bled from the jugular vein on 3 occasions prior to dosing and at 18 hr, 2, 7 and 14 days thereafter for serum analysis and hematology. Sera were analyzed for the activities of lactic dehydrogenase (LDH) and aspartate transaminase (AST) and for concentrations of total protein, albumin, globulin, bilirubin, cholesterol and urea by commercial kits (bio-Merieux Laboratory Reagents and Products, France). Blood samples were examined for packed cell volume (PCV), hemoglobin concentration (Hb), red blood cell (RBC), white blood cell (WBC), differential WBC counts, mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) by Cell Dyn R3500 (Abbot Diagnostics, IL, USA).

Statistical Analysis

Statistical significance was assessed by one-way ANOVA followed by Neuman-Keuls multiple comparison test (Snedecor and Cochran, 1976).

Results

Course of Toxicity

The dosing schedule and survival times of the N. oleander-poisoned sheep are given in Table 1. The control sheep in group 1 remained clinically healthy during the experiment and were humanely slaughtered on day 14. Sheep in groups 2 and 3 showed signs of poisoning within 3 hr which included restlessness, chewing movements of the jaws, moaning, salivation, ruminal bloat, frequent urination, incoordination of movements, paresis of the limbs and recumbency. Death occurred 4-24 hr after dosing. In group 4 sheep, signs of toxicosis appearing on day 3 were less marked and the 3 sheep died on days 3, 7 and 14, respectively.

Pathological Changes

Necropsy findings are summarized in Table 2. In groups 2 and 3, there was

congestion or hemorrhage in the brain, lungs, heart, liver, gall bladder, kidneys, urinary bladder, spleen, abomasum and intestines, pulmonary cyanosis and emphysema, catarrhal abomasitis and enteritis and fatty change or necrosis in the liver and kidneys. In group 4, congestion and hemorrhage in vital organs were less pronounced with obvious gelatinization of the renal pelvis and mesentry. The microscopic changes were similar but varied in extent and distribution. In groups 2 and 3, there was congestion of the pulmonary alveolar capillaries, hemorrhage into alveoli, alveolar emphysema and peribronchiolar lymphocytic infiltration. Fatty cytoplasmic vacuolation of the hepatocytes, congestion of the sinusoids and isolated lymphocytic nodules in the hepatic parenchyma occurred. The epithelial cells of many proximal convoluted tubules were degenerated and contained acidophilic homogeneous material in their lumens. The glomerular tufts were shrunken or necrotic and renal medullary rays were congested. The abomasal and intestinal lamina propria was congested and contained aggregates of lymphocytes. In group 4, hepatocyte fatty globules coalesced forming large-sized vacuoles (Figure 1) and renal tubular cells became necrotic. The control sheep (group 1) and the central and peripheral nervous systems of the test animals showed no significant changes.

[FIGURE 1 OMITTED]

Laboratory Results

Assays of serum were not done from the sheep of group 2 because none survived more than 5 hr. Table 3 shows the increases (p<0.05-0.001) in the activities of LDH and AST, in concentrations of cholesterol, bilirubin and urea and the decreases (p<0.05-0.001) in total protein and albumin levels in group 3 after 18 hr of dosing and in group 4 within 14 days of dosing. However, total protein and albumin concentrations in group 4 did not change 18 hr after dosing.

Hematological changes are presented in Table 4. There were significant differences (p<0.05-0.01) in WBC, neutrophils and lymphocytes between the controls and test groups after 18 hr and 14 days of dosing. Values of RBC, Hb and PCV in group 4 within 14 days of dosing and those of MCV in group 3 after 18 hr of dosing were lower (p<0.05-0.01) than controls. Eosinophils, basophils and monocytes did not change.

Discussion

The present study revealed that N. oleander leaves are fatal to Najdi sheep between 4 and 24 hr when given in single oral doses of 0.25 g/kg body weight or above. The repeated daily oral dose of N. oleander leaves at 0.06 g/kg body weight caused toxic manifestations and death between 3 and 14 days. The features described here call attention to the possibility of N. oleander toxicosis in grazing ruminants particularly at times of drought. Many plants such as Ipomoea carnea and Rhazya stricta were found to be hazardous when fed to small ruminants in the fresh or dry state (Abu Damir et al., 1987; Adam, 1998).

The development of nervous signs may be attributed to cerebral hemorrhage and congestion. The significant increases in the activities of serum LDH and AST and concentrations of bilirubin, cholesterol but lowered serum albumin concentrations indicate hepatocyte dysfunction. Renal lesions were sufficiently severe to cause significant elevations in serum urea concentration. Our work and the studies of Mahmoud et al. (1979) and Mohamed and Adam (1990) suggested that serum urea determination is useful for assessing kidney disease in small ruminants. Dyspnea was probably due to pulmonary lesions or ruminal bloat or to both. The widespread vascular changes could be due to endotheliotoxicity. Jatropha curcas and Piper abyssinica seeds were found to produce endotheliotoxicity and enterohepatonephropathy in ruminants (Adam and Magzoub, 1975; Ali et al., 1997) and chicks (El Badwi et al., 1992).

In sheep receiving 0.06 g dried N. oleander leaves/kg body weight/day, the presence of gelatinous material in the renal pelvis and mesentry indicates loss of condition and oxidation of the body reserve. In these animals, the alteration in erythrocytic series without effect on MCV or MCHC indicated normocytic normochromic anemia. The increase in lymphocytes was accompanied by lymphocytic infiltration in vital organs. The isolation and characterization of the active constituents in N. oleander leaves are in progress to elucidate the modes of action.

Acknowledgments

We wish to thank the General Directorate of Research Grants Programs, King Abdul Aziz City for Science and Technology, Riyadh, for financial support, Mr. E. E. El Mahi and Mr. N. Abdel Razig for technical assistance.

References

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(2.) Abu Damir, H., S.E.I. Adam and G. Tartour. The effects of Ipomoea carnea on goats and sheep. Vet. Hum. Toxicol. 29: 316-319, 1987.

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(12.) Migahid, A.M. Flora of Saudi Arabia: Riyadh University Press, 1978.

(13.) Mohamed, O.S.A. and S.E.I. Adam. The toxicity of temic (aldicarb) in Nubian goats. Br. Vet. Jour. 146: 358-361, 1990.

(14.) Osterloh, J., S. Herold and S. Pond. Oleander interference in the digoxin radioimmunoassay in a fatal ingestion. Jour. Am. Med. Ass. 247: 596-597, 1982.

(15.) Snedecor, G.M. and W. Cochran. Statistical Methods, 6th ed. Iowa State University Press, Ames, 10, 1976.

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S.E.I. Adam (1) *, M.A. Al-Yahya (2) and A.H. Al-Farhan (3)

(1) College of Agriculture and Veterinary Medicine, King Saud University, P.O. Box 1482, Buraidah, Al-Qassim,

(2) College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh and

(3) College of Science, King Saud University, P.O. Box 2455, Riyadh, Saudi Arabia

* Corresponding author

(Accepted for publication January 18, 2001)

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