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Neuropathologic study of long term cyanide administration to goats
Food and Chemical Toxicology 40 (2002) 1693–1698 www.elsevier.com/locate/foodchemtox
Neuropathologic study of long term cyanide administration to goats B. Soto-Blanco, P.C. Maiorka, S.L. Go´rniak* Research Center for Veterinary Toxicology (CEPTOX), Department of Pathology, School of Veterinary Medicine, University of Sa˜o Paulo, Av. Prof. Orlando Marques de Paiva, 87, Sa˜o Paulo—SP, Brazil 05508-900, Brazil Accepted 21 March 2002
Abstract Cyanogenic glycosides, which release cyanide, are present in several plant species of high importance for animal production, such as cassava and sorghum. Several human neurological diseases have been associated with chronic cyanide exposure. On the other hand, these effects in ruminants are almost unknown. Thus, the objective of the present study was to determine the long-term lesions of the central nervous system (CNS) caused by daily administration of potassium cyanide (KCN) to goats. Thirty-four male goats were divided into five groups, respectively treated orally with 0 (control), 0.3, 0.6, 1.2 or 3.0 mg KCN/kg/day for 5 months. At the end of the experiment, the whole CNS of each animal was collected for histopathology and immunohistochemistry for apoptotic markers (BAX, BCl2 and CPP32) and for glial fibrillary acid protein (GFAP; vimentin). The results showed the presence of spheroids in the pons, medulla oblongata, and ventral horn of the spinal cord, gliosis and spongiosis in medulla oblongata, gliosis in the pons, and damaged Purkinje cells in the cerebellum from goats that received the higher cyanide dose. In goats from the 1.2 mg KCN/kg group we observed congestion and hemorrhage in the cerebellum, and spheroids in the spinal cord. Gliosis was confirmed by GFAP protein expression. Immunohistochemistry for apoptotic markers and typical apoptotic morphology suggested apoptosis did not participate in the pathogenesis of the observed lesions. Thus, chronic cyanide exposure can promote neurophatological lesions also in goats, and this species can be a useful ruminant model to study the neurotoxic effects of long-term cyanide exposure. # 2002 Published by Elsevier Science Ltd. Keywords: Cyanide; Cyanogenic plants; Cassava; Neurological disease; Neurotoxicology; Apoptosis; Goats; Small ruminants
1. Introduction Cyanide is a ubiquitous substance in the environment and exposure to it may occur in a number of industrial processes such as metal processing, electroplating, plastic and chemical synthesis, and waste discharge from these and other industrial processes can contain large amounts of cyanide (El Ghawabi et al., 1975; Poulton, 1983; Blanc et al., 1985; Dictor et al., 1997). Cyanogenic glycosides occur naturally in many plants and may be another source of free cyanide if hydrolyzed. Thus, cyanogenic glycosides are synthesized in a number of cultivated forage plants of high importance for human and animal nutrition such as cassava and sorghum
Abbreviations: CNS, central nervous system; DAB, 3,30 -diaminobenzidine tetrahydrochloride; GFAP, glial fibrillary acid protein; KCN, potassium cyanide; PBS, phosphate buffered saline; StrepABC, streptoavidinbiotin peroxidase complex. * Corresponding author. E-mail address: [email protected] (S.L. Go´rniak).
(Poulton, 1983). Furthermore, some drugs of medicinal importance, such as Laetrile (Moertel et al., 1981) and nitroprusside (Schulz, 1984), can release cyanide. The acute toxicity of cyanide is well known. In this toxic process, inhibition of cytochrome oxidase occurs, resulting in disruption of aerobic respiration. This kind of intoxication has been controlled in many countries with education, plant selection and governmental regulation of plant importation (Poulton, 1983). On the other hand, neuropathies have been ascribed to prolonged exposure to cyanide at low concentration. In humans, chronic and spontaneous degenerative diseases such as tropical ataxic neuropathy (Osuntokun, 1981) and spastic paraparesis or ‘‘konzo’’ (Tylleska¨r et al., 1995) have been associated with high cassava consumption. Furthermore, prolonged cyanide exposure through both tobacco smoking and cassava consumption may be implicated in the pathogenesis of ocular pathologies such as tobacco-alcohol amblyopia (Pettigrew and Fell, 1972; Potts, 1973; Solberg et al., 1998), retrobulbar neuropathy of pernicious anemia (Freeman, 1988),
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Leber’s hereditary optic neuropathy (Syme et al., 1983; Tsao et al., 1999), West Indian amblyopia (Crews, 1963; MacKenzie and Phillips, 1968), Jamaican optic neuropathy (Carroll, 1971), tropical amblyopia (Osuntokun and Osuntokun, 1971) and Cuban optic neuropathy (Tucker and Hedges, 1993; The Cuban Optic Neuropathy Field Investigation Team, 1995). Cyanide has also been associated with syndromes affecting the central nervous system (CNS) in animals. Thus, ataxia has been observed in sheep, cattle and horses grazing sorghum, and urinary incontinence has also been observed in horses (Adams et al., 1969; McKenzie and McMicking, 1977; Bradley et al., 1995). The exact mechanism responsible for neuronal and/or axonal degeneration during prolonged exposure to cyanide is still unknown. Few experimental animal models have been proposed to better understand this form of cyanide intoxication. In addition, there is no information in the literature regarding experimental cyanide intoxication in ruminants. Therefore, the objective of the present study was to determine the effects of prolonged potassium cyanide (KCN) administration on the CNS of goats.
serial slices containing cortex, hippocampus, caudal midbrain, pons, medulla oblongata, cerebellum and spinal cord. All fragments were embedded in paraffin and 5-mm sections were stained with hematoxylin and eosin (HE) and with Bulshowsky staining. Immunohistochemistry and nuclear staining were performed in fragments from control animals and from animals treated with the two higher KCN doses.
2. Material and methods
2.4. Immunohistochemistry for apoptotic proteins
2.1. Animals and diets
Immunostaining was done by the streptoavidin–biotin peroxidase complex (StrepABC) method with 3,30 -diaminobenzidine tetrahydrochloride (DAB) as the substrate. Paraffin-embedded, formalin-fixed tissues were sectioned at 5 mm, mounted on slides and fixed by heating to 60 C for 20 min. Sections were deparaffinized in xylene and rehydrated through decreasing concentrations of graded ethanol. Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxidase/phosphate buffered saline (PBS) for 8 min at room temperature. Slides were rinsed in PBS and incubated overnight at room temperature with the primary antibodies (BAX, BCl2 and CPP32, on 1:100 dilution in PBS). The slides were then rinsed in PBS and incubated with the secondary antibody (1:500 dilution in PBS) for 2 h at room temperature. The sections were rinsed in PBS and incubated in streptoavidin peroxidase 1231 conjugate (1:500 dilution in PBS) for 2 h at room temperature. The peroxidase reaction was visualized with 0.05% DBA (Sigma) and 0.01% hydrogen peroxide and the material was rinsed in PBS and counterstained in Mayer’s hematoxylin. Negative controls were done by the same methodology but without use of the primary antibodies in order to confirm absence of unspecific bindings.
Thirty-four male crossbred Alpine-Saanen goats, 30– 45 days old, were used. The animals were fed Napier grass (Pennisetum purpureum schum) and common commercial ration for growing goats; Napier grass and water were given ad lib, and the amount of ration was 100 g per animal daily. For the first 3 months of the experiment, 600 ml of milk were given daily twice a day. The milk was from healthy cows and contained no pathogenic agents affecting goats. 2.2. Experimental design Goats were divided into five groups, four experimental and one control group. The experimental groups were dosed with 0.3 (seven animals), 0.6 (six animals), 1.2 (seven animals) or 3.0 (eight animals) mg of KCN (Merck, Germany)/kg of body weight/day, half administered in the morning and the other half in the afternoon. The control group (six animals) was not exposed to cyanide. KCN was administered through the milk for the first 3 months and orally after weaning, and control animals were similarly treated with water. Cyanide administration through milk or orally occurred at 07:30–08:00 h and at 16:30–17:00 h. One day after the last KCN administration, all goats were killed. The whole CNS was collected for histopathological study, fixed and stored in 10% buffered formalin. After complete fixation, the CNS was cut into
2.3. Antibodies Rabbit polyclonal antibodies against BAX, BCl2 and CPP32 (also designated as Yama, apopain and caspase3) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and polyclonal rabbit antibodies against cow glial fibrillary acidic protein (GFAP) and the monoclonal mouse antibody against vimentin (clone V9, purified from porcine eye lens) were obtained from DAKO A/S (Denmark). Biotinylated goat antirabbit immunoglobulins obtained from Sigma (St Louis, MO, USA) were employed as secondary antibodies. The use of goat antibodies as secondary ones did not produce unspecific reactions.
2.5. Nuclear fluorescent staining—Hoescht dye In order to evaluate the typical apoptotic nuclear features the nucleus was submitted to selective staining.
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Hoechst dye is a fluorescent DNA stain that intercalates in the A-T regions of DNA. Thus, Hoechst dye was used to improve the analysis of the characteristic nuclear morphology of apoptosis. Sections of 5 mm were mounted on slides and deparaffinized in xylene, rehydrated through decreasing concentrations of graded ethanol, and rinsed in PBS. The slides were then stained with Hoechst dye (1.0 mg/ml) for 10 min and rinsed briefly in PBS. The nucleoli were evaluated by fluorescence microscopy. 2.6. Glial-acidic fibrillary protein (GFAP) and vimentin immunostaining Immunostaining was performed by the StrepABC method. Primary antibodies against GFAP (dilution 1:1000) and against vimentin (dilution 1:1000) were used. Biotinylated goat antibody to mouse/rabbit immunoglobulins was employed as secondary antibody (dilution 1:400). The peroxidase reaction was visualized with 0.05% 3,30 -diaminobenzidine (Sigma) and 0.01% hydrogen peroxide. Negative controls were done by the same methodology but without use of the primary antibodies in order to confirm absence of unspecific bindings.
Plate 1. Grey matter of the spinal cord of a goat treated with 1.2 mg KCN/kg for 5 months showing axonal swelling in a transversal section. HE, 360.
2.7. Evaluation of stained slides BAX, BCl2, CPP32, GFAP and vimentin protein expression was assessed in terms of abundance and anatomic location of cells expressing these markers and the relationship between marker expression and particular morphologic aspect.
3. Results Clinical evaluation revealed no alteration in any animal tested except one from 3.0 mg KCN/kg group, which showed generalized muscular tremors and ataxia, but not convulsions, immediately after KCN administration. These symptoms were transient and lasted about 30 s and appeared at days 121–123 of the experiment. The histopathological study revealed CNS lesions in animals receiving the highest doses of KCN (1.2 and 3.0 mg KCN/kg). Thus, CNS sections from the 1.2 mg KCN/kg group showed congestion, hemorrhage and gliosis on the cerebellum, focal congestion and hemorrhage, gliosis, spheroids and axonal swelling on the grey matter of the spinal cord (Plate 1), and focal congestion, hemorrhage and gliosis in the pons. In animals that received 3.0 mg KCN/kg, damage and loss of several Purkinje cells were clearly visible in the cerebellum (Plates 2 and 3) and phagocitosis by mononuclear cells was evident in some of them. Spheroids, gliosis and spongiosis were observed in the pons, and spheroids, axonal swelling, gliosis, spongiosis and ghost cells were observed in the medulla oblongata. Spheroids were also
Plate 2. Cerebellum of a goat that received 3.0 mg KCN/kg for 5 months. Note the reduced number of Purkinje cells. HE, 40.
Plate 3. Cerebellum of a goat from the 3.0 mg KCN/kg group showing damaged Purkinje cells. HE, 360.
observed in the ventral horn of the spinal cord (Plate 4). The axonal spheroids were observed in both H&E and Bulshowsky stained sections, but they were better evidenced by the last staining.
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immunostaining for apoptosis presented the typical morphology or the nuclear appearance evidenced by Hoescht dye. Thus, neurons in apoptosis were not present in the material studied.
4. Discussion
Plate 4. Spheroid in the ventral horn of the spinal cord of a goat that received 3.0 mg KCN/kg. Bulshowsky, 360.
GFAP immunostaining was employed to confirm the gliosis observed by histology. Glial changes affected mostly astrocytes. Neither microglial nor oligodendroglial changes were detected. The staining intensity, but not the number, of GFAP-positive astrocytes was increased in the medulla oblongata and pons from goat treated with 3.0 mg KCN/kg. These astrocytes were immunomorphologically characterized by enlarged cell bodies, long branched cell processes and increased GFAP immunoreactivity (Plate 5). GFAP-negative control sections showed no positive reaction. In control and experimental animals, vimentin was detected in endothelial and leptomeningeal cells, and rarely in astrocytes. Vimentin-negative control sections showed no positive reaction. The results concerning apoptosis markers showed a total absence of BCl2 and CPP32 expression in all fragments evaluated. BAX reactivity was seldom observed in neurons of the granular portion of the cerebellum from both control and experimental animals. Examination of the characteristic apoptotic morphology was used to confirm this mode of cell death. No cells showing
Plate 5. Gliosis in the pons of a goat treated with 3.0 mg KCN/kg for 5 months. GFAP immunostaining, 240.
In humans, tropical ataxic neuropathy is classically characterized by bilateral primary optic atrophy, bilateral perceptive deafness, myelopathy and peripheral neuropathy (Osuntokun, 1981; Njoh, 1990). Patients suffering from ‘‘konzo’’ present isolated, non-progressive, spastic paraparesis of abrupt onset (Tylleska¨r et al., 1991). Furthermore, patients with tobacco-alcohol amblyopia, Leber’s hereditary optic atrophy, retrobulbar neuropathy of pernicious anemia and other ocular pathologies present failure of central vision (Syme et al., 1983; Freeman, 1988; van Heijst et al., 1994; Solberg et al., 1998; Tsao et al., 1999). In field studies conducted on horses (Adams et al., 1969) and cattle (McKenzie and McMicking, 1977) fed sorghum, mainly posterior ataxia and urinary incontinence were detected. Furthermore, horses and cattle could develop cystitis as a complication of urinary incontinence. Lambs grazing sorghum developed neurological signs consisting of knuckling of the fetlocks, weakness, stumbling and falling down with inability to rise, opisthotomos, head shaking, and muscle tremors (Bradley et al., 1995). In the present study, no clinical manifestation of the intoxication was observed, with only one goat from the KCN 3.0 mg/kg/day group showing transient generalized tremors and ataxia immediately after cyanide administration, which lasted less than 1 min. However, these symptoms appeared 4 months after the beginning of the experiment and were observed only for 3 consecutive days (days 121–123). It is difficult to understand the reason for the late and temporary manifestation of neurological signs in our goat, but it was definitely not a mistake in KCN dosing. It is feasible to suppose these symptoms are not related to chronic cyanide intoxication as the pathologies above described for both humans and animals. The single goat showing symptoms possibly presented temporarily exhaustion of the mechanism of cyanide detoxification. Effects of acute toxicity appearing after days of receiving cyanide or cyanogenic glycosides were also verified in three following studies conducted with goats in our laboratory (unpublished data), and another conducted with sheep (Juan Villalba and Frederick Provenza, personal communication). A key step in cyanide detoxification is catalyzed by rhodanese as cyanide is irreversibly bound with sulphur from compounds such thiosulphate and polythionates, producing thiocyanate. Although rhodanese is recycled and large amounts of cyanide can be metabolized, both cyanide and sulphite
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can inhibit rhodanese activity in the absence of thiosulphate (Bhatt and Linnell, 1987). Thus, the endogenous sulphur pool could be depleted, partially impairing cyanide detoxification. Unfortunately, histopathological examinations from humans chronically exposed to cyanide are lacking. On the other hand, some natural cases and experimental reproduction in animals of this exposure are available in the literature. Thus, spheroids were found in the white matter of the spinal cord, mostly in the ventral funiculi, and in the cerebellar peduncles of horses (Adams et al., 1969) and cattle (McKenzie and McMicking, 1977) fed sorghum. In lambs grazing the same forage, the predominant lesions found were formation of spheroids in the brain stem, cerebellum and ventral horn grey matter of the spinal cord, and mild gliosis (Bradley et al., 1995). In experimental studies on rats receiving KCN, neuronal damage was detected in the hippocampus, cortex and Purkinje cells of the cerebellum (Smith et al., 1963), and vacuolation and gliosis were observed in the spinal cord white matter (Philbrick et al., 1979). Similar to these earlier studies, the present data showed the occurrence of spheroids in the pons, medulla oblongata and ventral horn of the spinal cord, gliosis and spongiosis in the medulla oblongata, gliosis in pons, and damaged Purkinje cells in the cerebellum from goats treated with KCN. Damage of Purkinje neurons was possibly caused by cellular hypoxia promoted by cyanide once it also occurs in other causes of hypoxia (Cervo´s-Navarro et al., 1991). In goats that received the higher KCN dose, GFAP-positive astrocytes showing increased staining intensity did not express vimentin. It has been reported vimentin expression with or without concomitant GFAP expression may reflect a reactive or degenerative change in astrocytes and this reaction could be interpreted as a transient reversion to an immature phenotype (Arnold et al., 1996; Gaedke et al., 1999). However, in the present study vimentin did not show immunohistologically detectable expression. Thus, we may assume that in chronic cyanide intoxication astrocytes were branched but not reactive. Cyanide-induced apoptosis was described in vitro in differentiated, but not undifferentiated, pheochromocytoma clonal cell line (PC12) (Mills et al., 1996) as well as in vivo in neurons of the motor cortex from mice (Mills et al., 1999). Upon cyanide exposure, differentiated PC12 cells undergo a transcription-dependent programmed cell death characterized by endonuclease activation, DNA fragmentation, and the typical morphological changes of apoptosis (Mills et al., 1996). In mice, cyanide administration promoted apoptosis in neurons from the substantia nigra, whereas cortical neurons presented necrosis (Mills et al., 1999). However, apoptosis appeared not to be involved in the pathogenesis of the lesions observed in goats chronically receiving cyanide. A possible explanation for this discrepancy is
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that apoptosis could be present at the beginning of the formation of the lesions, and was no longer present thereafter. Evidence supporting this possibility was provided by the study of Mills et al. (1999), who observed a peak of apoptosis frequency 3 days after cyanide administration, followed by a reduction of apoptosis intensity. Furthermore, the same authors suggested cyanide could produce necrosis or apoptosis according to different neuronal populations. In summary, we have demonstrated the neurotoxic effects promoted by long-term cyanide exposure in goats, and we have proposed a new animal model to study cyanide toxicity. Thus, a broader spectrum of information can be obtained from the association of the present model with those currently available.
Acknowledgements The authors wish to thank Mrs. Silvia C.S. Oloris and Mrs. Katia N. Ebina for technical assistance in the immunohistochemical procedures. The language correction by Elettra Greene is also acknowledged. This work is part of the Master’s thesis presented by Benito Soto-Blanco to the Department of Pathology, School of Veterinary Medicine, University of Sa˜o Paulo, Brazil, supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP).
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El Ghawabi, S.H., Gaafar, M.A., El Saharti, A.A., Ahmed, S.H., Malash, K.K., Fares, R., 1975. Chronic cyanide exposure: a clinical, radioisotope, and laboratory study. British Journal of Industrial Medicine 32, 215–219. Freeman, A.G., 1988. Optic neuropathy and chronic cyanide intoxication: a review. Journal of the Royal Society of Medicine 81, 103–106. Gaedke, K., Zurbriggen, A., Baumga¨rtner, W., 1999. Lack of correlation between virus nucleoprotein and mRNA expression and the inflammatory response in demyelinating distemper encephalitis indicates a biphasic disease process. European Journal of Veterinary Pathology 5, 9–20. MacKenzie, A.D., Phillips, C.I., 1968. West Indian amblyopia. Brain 91, 249–260. McKenzie, R.A., McMicking, L.I., 1977. Ataxia and urinary incontinence in cattle grazing sorghum. Australian Veterinary Journal 53, 496–497. Mills, E.M., Gunasekar, P.G., Li, L., Borowitz, J.L., Isom, G.E., 1999. Differential susceptibility of brain areas to cyanide involves different modes of cell death. Toxicology and Applied Pharmacology 156, 6–16. Mills, E.M., Gunasekar, P.G., Pavlakovic, G., Isom, G.E., 1996. Cyanide-induced apoptosis and oxidative stress in differentiated PC12 cells. Journal of Neurochemistry 67, 1039–1046. Moertel, C.G., Ames, M.M., Kovach, J.S., Moyer, T.P., Rubin, J.R., Tinker, J.H., 1981. A pharmacological and toxicological study of amygdalin. Journal of the American Medical Association 245, 591–594. Njoh, J., 1990. Tropical ataxic neuropathy in liberians. Tropical and Geographical Medicine 42, 92–94. Osuntokun, B.O., 1981. Cassava diet, chronic cyanide intoxication and neuropathy in the Nigerian Africans. World Review on Nutrition and Dietetics 36, 141–173. Osuntokun, B.O., Osuntokun, O., 1971. Tropical amblyopia in Nigerians. American Journal of Ophthalmology 72, 708–716. Pettigrew, A.R., Fell, G.S., 1972. Simplified colorimetric determination of thiocyanate in biological fluids and its application to investigation of the toxic amblyopias. Clinical Chemistry 18, 996–1000. Philbrick, D.J., Hopkins, J.B., Hill, D.C., Alexander, J.C., Thomson, R.G., 1979. Effects of prolonged cyanide and thiocyanate feeding in rats. Journal of Toxicology and Environmental Health 5, 579–592.
Potts, A.M., 1973. Tobacco amblyopia. Survey on Ophthalmology 17, 313–339. Poulton, J.E., 1983. Cyanogenic compounds in plants and their toxic effects. In: Keeler, R.F., Tu, A.T. (Eds.), Handbook of Natural Toxins, Vol. 1 (Plant and Fungal Toxins). Marcel Dekker, New York, pp. 117–157. Schulz, V., 1984. Clinical pharmacokinetics of nitroprusside, cyanide, thiosulphate and thiocyanate. Clinical Pharmacokinetics 9, 239–251. Smith, A.D.M., Duckett, S., Waters, A.H., 1963. Neuropathological changes in chronic cyanide intoxication. Nature 200, 179–181. Solberg, Y., Rosner, M., Belkin, M., 1998. The association between cigarette smoking and ocular diseases. Survey on Ophthalmology 42, 535–547. Syme, I.G., Bronte-Stewart, J., Foulds, W.S., McClure, E., Logan, R., Stansfield, M., 1983. Clinical and biochemical findings in Leber’s hereditary optic atrophy. Transactions of the Ophthalmologic Society of the United Kingdom 103, 556–559. Tsao, K., Aiken, P.A., Johns, D.R., 1999. Smoking as an aetiological factor in a pedigree with Leber’s hereditary optic neuropathy. British Journal of Ophthalmology 83, 577–581. The Cuban Optic Neuropathy Field Investigation Team, 1995. Epidemic optic neuropathy in Cuba—clinical characterisation and risk factors. New England Journal of Medicine 333, 1176–1182. Tucker, K., Hedges, T.R., 1993. Food shortages and an epidemic of optic and peripheral neuropathy in Cuba. Nutrition Reviews 51, 349–357. Tylleska¨r, T., Banea, M., Bikangi, N., Fresco, L., Persson, L.A˚., Rosling, H., 1991. Epidemiological evidence from Zaire for a dietary etiology of konzo, an upper motor neuron disease. Bulletin of World Health Organization 69:5, 581–589. Tylleska¨r, T., Banea, M., Bikangi, N., Nahimana, G., Persson, L.A˚., Rosling, H., 1995. Dietary determinants of a non-progressive spastic paraparesis (Konzo): a case-referent study in a high incidence area of Zaire. International Journal of Epidemiology 24, 949– 956. van Heijst, A.N., Maes, R.A., Mtanda, A.T., Chuwa, L.M., Rwiza, H.T., Moshi, N.H., 1994. Chronic cyanide poisoning in relation to blindness and tropical neuropathy. Journal of Toxicology Clinical Toxicology 32, 549–556.
Neuropathologic study of long term cyanide administration to goats B. Soto-Blanco, P.C. Maiorka, S.L. Go´rniak* Research Center for Veterinary Toxicology (CEPTOX), Department of Pathology, School of Veterinary Medicine, University of Sa˜o Paulo, Av. Prof. Orlando Marques de Paiva, 87, Sa˜o Paulo—SP, Brazil 05508-900, Brazil Accepted 21 March 2002
Abstract Cyanogenic glycosides, which release cyanide, are present in several plant species of high importance for animal production, such as cassava and sorghum. Several human neurological diseases have been associated with chronic cyanide exposure. On the other hand, these effects in ruminants are almost unknown. Thus, the objective of the present study was to determine the long-term lesions of the central nervous system (CNS) caused by daily administration of potassium cyanide (KCN) to goats. Thirty-four male goats were divided into five groups, respectively treated orally with 0 (control), 0.3, 0.6, 1.2 or 3.0 mg KCN/kg/day for 5 months. At the end of the experiment, the whole CNS of each animal was collected for histopathology and immunohistochemistry for apoptotic markers (BAX, BCl2 and CPP32) and for glial fibrillary acid protein (GFAP; vimentin). The results showed the presence of spheroids in the pons, medulla oblongata, and ventral horn of the spinal cord, gliosis and spongiosis in medulla oblongata, gliosis in the pons, and damaged Purkinje cells in the cerebellum from goats that received the higher cyanide dose. In goats from the 1.2 mg KCN/kg group we observed congestion and hemorrhage in the cerebellum, and spheroids in the spinal cord. Gliosis was confirmed by GFAP protein expression. Immunohistochemistry for apoptotic markers and typical apoptotic morphology suggested apoptosis did not participate in the pathogenesis of the observed lesions. Thus, chronic cyanide exposure can promote neurophatological lesions also in goats, and this species can be a useful ruminant model to study the neurotoxic effects of long-term cyanide exposure. # 2002 Published by Elsevier Science Ltd. Keywords: Cyanide; Cyanogenic plants; Cassava; Neurological disease; Neurotoxicology; Apoptosis; Goats; Small ruminants
1. Introduction Cyanide is a ubiquitous substance in the environment and exposure to it may occur in a number of industrial processes such as metal processing, electroplating, plastic and chemical synthesis, and waste discharge from these and other industrial processes can contain large amounts of cyanide (El Ghawabi et al., 1975; Poulton, 1983; Blanc et al., 1985; Dictor et al., 1997). Cyanogenic glycosides occur naturally in many plants and may be another source of free cyanide if hydrolyzed. Thus, cyanogenic glycosides are synthesized in a number of cultivated forage plants of high importance for human and animal nutrition such as cassava and sorghum
Abbreviations: CNS, central nervous system; DAB, 3,30 -diaminobenzidine tetrahydrochloride; GFAP, glial fibrillary acid protein; KCN, potassium cyanide; PBS, phosphate buffered saline; StrepABC, streptoavidinbiotin peroxidase complex. * Corresponding author. E-mail address: [email protected] (S.L. Go´rniak).
(Poulton, 1983). Furthermore, some drugs of medicinal importance, such as Laetrile (Moertel et al., 1981) and nitroprusside (Schulz, 1984), can release cyanide. The acute toxicity of cyanide is well known. In this toxic process, inhibition of cytochrome oxidase occurs, resulting in disruption of aerobic respiration. This kind of intoxication has been controlled in many countries with education, plant selection and governmental regulation of plant importation (Poulton, 1983). On the other hand, neuropathies have been ascribed to prolonged exposure to cyanide at low concentration. In humans, chronic and spontaneous degenerative diseases such as tropical ataxic neuropathy (Osuntokun, 1981) and spastic paraparesis or ‘‘konzo’’ (Tylleska¨r et al., 1995) have been associated with high cassava consumption. Furthermore, prolonged cyanide exposure through both tobacco smoking and cassava consumption may be implicated in the pathogenesis of ocular pathologies such as tobacco-alcohol amblyopia (Pettigrew and Fell, 1972; Potts, 1973; Solberg et al., 1998), retrobulbar neuropathy of pernicious anemia (Freeman, 1988),
0278-6915/02/$ - see front matter # 2002 Published by Elsevier Science Ltd. PII: S0278-6915(02)00151-5
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Leber’s hereditary optic neuropathy (Syme et al., 1983; Tsao et al., 1999), West Indian amblyopia (Crews, 1963; MacKenzie and Phillips, 1968), Jamaican optic neuropathy (Carroll, 1971), tropical amblyopia (Osuntokun and Osuntokun, 1971) and Cuban optic neuropathy (Tucker and Hedges, 1993; The Cuban Optic Neuropathy Field Investigation Team, 1995). Cyanide has also been associated with syndromes affecting the central nervous system (CNS) in animals. Thus, ataxia has been observed in sheep, cattle and horses grazing sorghum, and urinary incontinence has also been observed in horses (Adams et al., 1969; McKenzie and McMicking, 1977; Bradley et al., 1995). The exact mechanism responsible for neuronal and/or axonal degeneration during prolonged exposure to cyanide is still unknown. Few experimental animal models have been proposed to better understand this form of cyanide intoxication. In addition, there is no information in the literature regarding experimental cyanide intoxication in ruminants. Therefore, the objective of the present study was to determine the effects of prolonged potassium cyanide (KCN) administration on the CNS of goats.
serial slices containing cortex, hippocampus, caudal midbrain, pons, medulla oblongata, cerebellum and spinal cord. All fragments were embedded in paraffin and 5-mm sections were stained with hematoxylin and eosin (HE) and with Bulshowsky staining. Immunohistochemistry and nuclear staining were performed in fragments from control animals and from animals treated with the two higher KCN doses.
2. Material and methods
2.4. Immunohistochemistry for apoptotic proteins
2.1. Animals and diets
Immunostaining was done by the streptoavidin–biotin peroxidase complex (StrepABC) method with 3,30 -diaminobenzidine tetrahydrochloride (DAB) as the substrate. Paraffin-embedded, formalin-fixed tissues were sectioned at 5 mm, mounted on slides and fixed by heating to 60 C for 20 min. Sections were deparaffinized in xylene and rehydrated through decreasing concentrations of graded ethanol. Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxidase/phosphate buffered saline (PBS) for 8 min at room temperature. Slides were rinsed in PBS and incubated overnight at room temperature with the primary antibodies (BAX, BCl2 and CPP32, on 1:100 dilution in PBS). The slides were then rinsed in PBS and incubated with the secondary antibody (1:500 dilution in PBS) for 2 h at room temperature. The sections were rinsed in PBS and incubated in streptoavidin peroxidase 1231 conjugate (1:500 dilution in PBS) for 2 h at room temperature. The peroxidase reaction was visualized with 0.05% DBA (Sigma) and 0.01% hydrogen peroxide and the material was rinsed in PBS and counterstained in Mayer’s hematoxylin. Negative controls were done by the same methodology but without use of the primary antibodies in order to confirm absence of unspecific bindings.
Thirty-four male crossbred Alpine-Saanen goats, 30– 45 days old, were used. The animals were fed Napier grass (Pennisetum purpureum schum) and common commercial ration for growing goats; Napier grass and water were given ad lib, and the amount of ration was 100 g per animal daily. For the first 3 months of the experiment, 600 ml of milk were given daily twice a day. The milk was from healthy cows and contained no pathogenic agents affecting goats. 2.2. Experimental design Goats were divided into five groups, four experimental and one control group. The experimental groups were dosed with 0.3 (seven animals), 0.6 (six animals), 1.2 (seven animals) or 3.0 (eight animals) mg of KCN (Merck, Germany)/kg of body weight/day, half administered in the morning and the other half in the afternoon. The control group (six animals) was not exposed to cyanide. KCN was administered through the milk for the first 3 months and orally after weaning, and control animals were similarly treated with water. Cyanide administration through milk or orally occurred at 07:30–08:00 h and at 16:30–17:00 h. One day after the last KCN administration, all goats were killed. The whole CNS was collected for histopathological study, fixed and stored in 10% buffered formalin. After complete fixation, the CNS was cut into
2.3. Antibodies Rabbit polyclonal antibodies against BAX, BCl2 and CPP32 (also designated as Yama, apopain and caspase3) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and polyclonal rabbit antibodies against cow glial fibrillary acidic protein (GFAP) and the monoclonal mouse antibody against vimentin (clone V9, purified from porcine eye lens) were obtained from DAKO A/S (Denmark). Biotinylated goat antirabbit immunoglobulins obtained from Sigma (St Louis, MO, USA) were employed as secondary antibodies. The use of goat antibodies as secondary ones did not produce unspecific reactions.
2.5. Nuclear fluorescent staining—Hoescht dye In order to evaluate the typical apoptotic nuclear features the nucleus was submitted to selective staining.
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Hoechst dye is a fluorescent DNA stain that intercalates in the A-T regions of DNA. Thus, Hoechst dye was used to improve the analysis of the characteristic nuclear morphology of apoptosis. Sections of 5 mm were mounted on slides and deparaffinized in xylene, rehydrated through decreasing concentrations of graded ethanol, and rinsed in PBS. The slides were then stained with Hoechst dye (1.0 mg/ml) for 10 min and rinsed briefly in PBS. The nucleoli were evaluated by fluorescence microscopy. 2.6. Glial-acidic fibrillary protein (GFAP) and vimentin immunostaining Immunostaining was performed by the StrepABC method. Primary antibodies against GFAP (dilution 1:1000) and against vimentin (dilution 1:1000) were used. Biotinylated goat antibody to mouse/rabbit immunoglobulins was employed as secondary antibody (dilution 1:400). The peroxidase reaction was visualized with 0.05% 3,30 -diaminobenzidine (Sigma) and 0.01% hydrogen peroxide. Negative controls were done by the same methodology but without use of the primary antibodies in order to confirm absence of unspecific bindings.
Plate 1. Grey matter of the spinal cord of a goat treated with 1.2 mg KCN/kg for 5 months showing axonal swelling in a transversal section. HE, 360.
2.7. Evaluation of stained slides BAX, BCl2, CPP32, GFAP and vimentin protein expression was assessed in terms of abundance and anatomic location of cells expressing these markers and the relationship between marker expression and particular morphologic aspect.
3. Results Clinical evaluation revealed no alteration in any animal tested except one from 3.0 mg KCN/kg group, which showed generalized muscular tremors and ataxia, but not convulsions, immediately after KCN administration. These symptoms were transient and lasted about 30 s and appeared at days 121–123 of the experiment. The histopathological study revealed CNS lesions in animals receiving the highest doses of KCN (1.2 and 3.0 mg KCN/kg). Thus, CNS sections from the 1.2 mg KCN/kg group showed congestion, hemorrhage and gliosis on the cerebellum, focal congestion and hemorrhage, gliosis, spheroids and axonal swelling on the grey matter of the spinal cord (Plate 1), and focal congestion, hemorrhage and gliosis in the pons. In animals that received 3.0 mg KCN/kg, damage and loss of several Purkinje cells were clearly visible in the cerebellum (Plates 2 and 3) and phagocitosis by mononuclear cells was evident in some of them. Spheroids, gliosis and spongiosis were observed in the pons, and spheroids, axonal swelling, gliosis, spongiosis and ghost cells were observed in the medulla oblongata. Spheroids were also
Plate 2. Cerebellum of a goat that received 3.0 mg KCN/kg for 5 months. Note the reduced number of Purkinje cells. HE, 40.
Plate 3. Cerebellum of a goat from the 3.0 mg KCN/kg group showing damaged Purkinje cells. HE, 360.
observed in the ventral horn of the spinal cord (Plate 4). The axonal spheroids were observed in both H&E and Bulshowsky stained sections, but they were better evidenced by the last staining.
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immunostaining for apoptosis presented the typical morphology or the nuclear appearance evidenced by Hoescht dye. Thus, neurons in apoptosis were not present in the material studied.
4. Discussion
Plate 4. Spheroid in the ventral horn of the spinal cord of a goat that received 3.0 mg KCN/kg. Bulshowsky, 360.
GFAP immunostaining was employed to confirm the gliosis observed by histology. Glial changes affected mostly astrocytes. Neither microglial nor oligodendroglial changes were detected. The staining intensity, but not the number, of GFAP-positive astrocytes was increased in the medulla oblongata and pons from goat treated with 3.0 mg KCN/kg. These astrocytes were immunomorphologically characterized by enlarged cell bodies, long branched cell processes and increased GFAP immunoreactivity (Plate 5). GFAP-negative control sections showed no positive reaction. In control and experimental animals, vimentin was detected in endothelial and leptomeningeal cells, and rarely in astrocytes. Vimentin-negative control sections showed no positive reaction. The results concerning apoptosis markers showed a total absence of BCl2 and CPP32 expression in all fragments evaluated. BAX reactivity was seldom observed in neurons of the granular portion of the cerebellum from both control and experimental animals. Examination of the characteristic apoptotic morphology was used to confirm this mode of cell death. No cells showing
Plate 5. Gliosis in the pons of a goat treated with 3.0 mg KCN/kg for 5 months. GFAP immunostaining, 240.
In humans, tropical ataxic neuropathy is classically characterized by bilateral primary optic atrophy, bilateral perceptive deafness, myelopathy and peripheral neuropathy (Osuntokun, 1981; Njoh, 1990). Patients suffering from ‘‘konzo’’ present isolated, non-progressive, spastic paraparesis of abrupt onset (Tylleska¨r et al., 1991). Furthermore, patients with tobacco-alcohol amblyopia, Leber’s hereditary optic atrophy, retrobulbar neuropathy of pernicious anemia and other ocular pathologies present failure of central vision (Syme et al., 1983; Freeman, 1988; van Heijst et al., 1994; Solberg et al., 1998; Tsao et al., 1999). In field studies conducted on horses (Adams et al., 1969) and cattle (McKenzie and McMicking, 1977) fed sorghum, mainly posterior ataxia and urinary incontinence were detected. Furthermore, horses and cattle could develop cystitis as a complication of urinary incontinence. Lambs grazing sorghum developed neurological signs consisting of knuckling of the fetlocks, weakness, stumbling and falling down with inability to rise, opisthotomos, head shaking, and muscle tremors (Bradley et al., 1995). In the present study, no clinical manifestation of the intoxication was observed, with only one goat from the KCN 3.0 mg/kg/day group showing transient generalized tremors and ataxia immediately after cyanide administration, which lasted less than 1 min. However, these symptoms appeared 4 months after the beginning of the experiment and were observed only for 3 consecutive days (days 121–123). It is difficult to understand the reason for the late and temporary manifestation of neurological signs in our goat, but it was definitely not a mistake in KCN dosing. It is feasible to suppose these symptoms are not related to chronic cyanide intoxication as the pathologies above described for both humans and animals. The single goat showing symptoms possibly presented temporarily exhaustion of the mechanism of cyanide detoxification. Effects of acute toxicity appearing after days of receiving cyanide or cyanogenic glycosides were also verified in three following studies conducted with goats in our laboratory (unpublished data), and another conducted with sheep (Juan Villalba and Frederick Provenza, personal communication). A key step in cyanide detoxification is catalyzed by rhodanese as cyanide is irreversibly bound with sulphur from compounds such thiosulphate and polythionates, producing thiocyanate. Although rhodanese is recycled and large amounts of cyanide can be metabolized, both cyanide and sulphite
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can inhibit rhodanese activity in the absence of thiosulphate (Bhatt and Linnell, 1987). Thus, the endogenous sulphur pool could be depleted, partially impairing cyanide detoxification. Unfortunately, histopathological examinations from humans chronically exposed to cyanide are lacking. On the other hand, some natural cases and experimental reproduction in animals of this exposure are available in the literature. Thus, spheroids were found in the white matter of the spinal cord, mostly in the ventral funiculi, and in the cerebellar peduncles of horses (Adams et al., 1969) and cattle (McKenzie and McMicking, 1977) fed sorghum. In lambs grazing the same forage, the predominant lesions found were formation of spheroids in the brain stem, cerebellum and ventral horn grey matter of the spinal cord, and mild gliosis (Bradley et al., 1995). In experimental studies on rats receiving KCN, neuronal damage was detected in the hippocampus, cortex and Purkinje cells of the cerebellum (Smith et al., 1963), and vacuolation and gliosis were observed in the spinal cord white matter (Philbrick et al., 1979). Similar to these earlier studies, the present data showed the occurrence of spheroids in the pons, medulla oblongata and ventral horn of the spinal cord, gliosis and spongiosis in the medulla oblongata, gliosis in pons, and damaged Purkinje cells in the cerebellum from goats treated with KCN. Damage of Purkinje neurons was possibly caused by cellular hypoxia promoted by cyanide once it also occurs in other causes of hypoxia (Cervo´s-Navarro et al., 1991). In goats that received the higher KCN dose, GFAP-positive astrocytes showing increased staining intensity did not express vimentin. It has been reported vimentin expression with or without concomitant GFAP expression may reflect a reactive or degenerative change in astrocytes and this reaction could be interpreted as a transient reversion to an immature phenotype (Arnold et al., 1996; Gaedke et al., 1999). However, in the present study vimentin did not show immunohistologically detectable expression. Thus, we may assume that in chronic cyanide intoxication astrocytes were branched but not reactive. Cyanide-induced apoptosis was described in vitro in differentiated, but not undifferentiated, pheochromocytoma clonal cell line (PC12) (Mills et al., 1996) as well as in vivo in neurons of the motor cortex from mice (Mills et al., 1999). Upon cyanide exposure, differentiated PC12 cells undergo a transcription-dependent programmed cell death characterized by endonuclease activation, DNA fragmentation, and the typical morphological changes of apoptosis (Mills et al., 1996). In mice, cyanide administration promoted apoptosis in neurons from the substantia nigra, whereas cortical neurons presented necrosis (Mills et al., 1999). However, apoptosis appeared not to be involved in the pathogenesis of the lesions observed in goats chronically receiving cyanide. A possible explanation for this discrepancy is
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that apoptosis could be present at the beginning of the formation of the lesions, and was no longer present thereafter. Evidence supporting this possibility was provided by the study of Mills et al. (1999), who observed a peak of apoptosis frequency 3 days after cyanide administration, followed by a reduction of apoptosis intensity. Furthermore, the same authors suggested cyanide could produce necrosis or apoptosis according to different neuronal populations. In summary, we have demonstrated the neurotoxic effects promoted by long-term cyanide exposure in goats, and we have proposed a new animal model to study cyanide toxicity. Thus, a broader spectrum of information can be obtained from the association of the present model with those currently available.
Acknowledgements The authors wish to thank Mrs. Silvia C.S. Oloris and Mrs. Katia N. Ebina for technical assistance in the immunohistochemical procedures. The language correction by Elettra Greene is also acknowledged. This work is part of the Master’s thesis presented by Benito Soto-Blanco to the Department of Pathology, School of Veterinary Medicine, University of Sa˜o Paulo, Brazil, supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP).
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