- Email: [email protected]
Effects of Long-Term Low-Dose Cyanide Administration to Rats
Ecotoxicology and Environmental Safety 53, 37}41 (2002) Environmental Research, Section B doi:10.1006/eesa.2002.2189
Effects of Long-Term Low-Dose Cyanide Administration to Rats1 Benito Soto-Blanco, Paulo CeH sar Marioka, and Silvana Lima GoH rniak Research Center for Veterinary Toxicology, Department of Pathology, School of Veterinary Medicine, University of Sa o Paulo, Brazil Received October 25, 2000
feeding have cyanogenic glycosides that release cyanide. The most important cyanogenic plant consumed by humans and animals worldwide is cassava (Manihot esculenta Crantz), which contains mainly linamarin as its cyanogenic glycoside (Poulton, 1983). With respect to the mechanism of action, inhibition of cellular respiration is produced by binding of cyanide to ferric iron in cytochrome oxidase, resulting in histotoxic cellular hypoxia or anoxia. Nevertheless, mammals have an e$cient mechanism of detoxi"cation of this poison through conversion to thiocyanate by rhodanase, a mitochondrial enzyme (Way, 1984). Acute toxicity has been extensively studied, but there is a paucity of information about chronic cyanide exposure. On the other hand, it has been assumed that the more widely distributed adverse e!ects of cyanide are due its chronic toxicity. Probably the most pathologic conditions attributable to prolonged exposure to cyanide are the neuropathies. Thus, degenerative diseases such as tropical ataxic neuropathy (TAN) (Osuntokun, 1981) and spastic paraparesis or &&konzo'' (TylleskaK r et al., 1995) have been associated with high cassava consumption. Furthermore, prolonged cyanide exposure through both tobacco smoke and cassava consumption may be implicated in the pathogenesis of ocular pathologies such as tobacco}alcohol amblyopia (Solberg et al., 1998), retrobulbar neuropathy of pernicious anemia (Freeman, 1988), Leber's hereditary optic neuropathy (Tsao et al., 1999), and Cuban optic neuropathy (Tucker and Hedges, 1993). Horses, cattle, and sheep grazing on cyanogenic Sorghum pastures developed neuronal disorders, especially ataxia (Adams et al., 1969; McKenzie and McMicking, 1977; Bradley et al., 1995). There are many studies reporting a correlation between cyanogenic plant ingestion and development of goiter in di!erent animal species (Ratnakumar and Rajan, 1992; Tewe et al., 1984; Gutzwiller, 1993) and humans (Gaitan et al., 1994). Goiter is related to the main product of transformation of cyanide, thiocyanate, which competes with iodide in the Na>/l\ symporter in the thyroid gland, consequently inhibiting the synthesis and clearance of thyroid hormones (Dohan et al., 2000).
Chronic exposure to cyanide has been associated with development of pancreatic diabetes, hypothyroidism, and several neurological diseases in both humans and animals. However, there is a limited number of experimental models for these pathologies. Thus, in the present study 0, 0.15, 0.3, or 0.6 mg KCN/kg body weight/day was administered for 3 months to 26 rats. On the last day, plasma samples were obtained for glucose, cholesterol, and thyroidal hormone measurement, and the pancreas, thyroids, and whole central nervous system were collected for histopathologic study. There were no signi5cant di4erence in plasma concentrations of the substances measured between groups, and no lesions were found in the pancrease or thyroid. The CNS of experimental animals revealed the presence of spheroids on the ventral horn of the spinal cord, neuron loss in the hippocampus, damaged Purkinje cells, and loss of cerebellar white matter. In conclusion, cyanide administration could promote neuropathological lesions in rats without a4ecting pancreas or thyroid gland metabolism. 2002 Elsevier Science (USA) Key Words: cyanide; tropical diabetes; hypothyroidism; goiter; neurotoxicity.
INTRODUCTION
Cyanide is a ubiquitous toxic ion in the environment. Exposure to this substance occurs through several sources. Cyanide is present in many industrial processes such as hardening of metals, electroplating, chemical synthesis, and gold extraction. Environmental pollution can be produced by waste discharges from these industries (Poulton, 1983; Boening and Chew, 1999). This ion is also found in smoke from tobacco, "res, and propulsion motors (Poulton, 1983). Some drugs, such as nitroprusside and laetrile, can release cyanide (Moertel et al., 1981; Schulz, 1984). Furthermore, a large number of plant species for human and animal This study was supported by a grant from FAPESP (Fundac,a o de Amparo a` Pesquisa do Estado de Sa o Paulo). To whom correspondence should be addressed at Research Center for Veterinary Toxicology (CEPTOX), Department of Pathology, School of Veterinary Medicine, University of Sa o Paulo, Av. Orlando Marques de Paiva 87, Sa o Paulo 05508-900, Brazil. E-mail: [email protected]. 37
0147-6513/02 $35.00 2002 Elsevier Science (USA) All rights reserved.
38
SOTO-BLANCO, MARIOKA, AND GOD RNIAK
Furthermore, the development of pancreatic diabetes, known as tropical diabetes, in humans has been associated with consumption of cyanogenic plants, especially cassava (McMillan and Geevarghese, 1979; Assan et al., 1988; Pitchumoni et al., 1988; Kamalu, 1995). McMillan and Geevarghese (1979) suggested that pancreatic diabetes could be produced by the action of cyanide release by cassava on the acinar portion of the pancreas, thus a!ecting the endocrine portion. However, no experimental model reproducing pancreatic diabetes by administration of cyanide itself is available in the literature. In spite of the widespread distribution of cyanide-containing sources and its possible implication as the etiology of various human and animal diseases, it has been di$cult to precisely assess the contribution of this substance to the clinical disease, since most of the evidence is based on "eld studies without controlled experimental conditions. Thus, the purpose of the present study was to determine the e!ects of long-term low-dose potassium cyanide (KCN) administration on the central nervous system (CNS) and thyroid and pancreatic metabolism, using rats as an animal model. MATERIAL AND METHODS
Used were 26 weanling male Wistar rats, 22 days old, weighing on average 44 g at the beginning of the experiment. The animals were kept in groups of 3 or 4 in 33;41-cm acrylic cages at 22}243C on a 10-h light} 14-h dark cycle (lights on at 7:00 AM) with free access to food (Nutrilab, Brazil) and water. The animals were randomly divided into four groups, dosed with 0 (7 animals), 0.15 (6 animals), 0.3 (6 animals), or 0.6 (7 animals) mg KCN/body weight/day by gavage every day for 3 months. These KCN doses are 1.5, 3.0, and 6.0% of the peroral LD for male rats (10 mg/kg) (Hayes, 1967). Cyanide was always administered at the same time of day (9:00 to 10:00 AM), and KCN solutions were prepared fresh every day. The health of the rats was checked daily, body weight was recorded twice a week to adjust the KCN dose, and food consumption was recorded on the same day as body weight. At the end of the experiment, blood samples were collected from the hepatic vein of rats under ether anesthesia with heparinized (Liquemine, Roche, Brazil) syringes (SR, Brazil) in order to obtain plasma. These samples were frozen at !103C until analysis. Plasma cholesterol and glucose levels were determined using a commercial kit (Laborlab, Brazil), and thyroxine (T4) and triiodothyronine (T3) levels were measured by radioimmunoassay (DPC). After blood collection, the animals were killed by transcardiac perfusion with 10% bu!ered formalin followed by Carnoy solution. Whole CNS, pancreas, and thyroid glands were collected for histopathologic study. After complete "xation, the CNS was cut into fragments containing cortex, hippocampus, brain stem (caudal midbrain, pons and
FIG. 1. Body weight gain of rats treated with di!erent KCN doses.
medulla oblongata), cerebellum, and spinal cord. CNS and thyroid gland fragments were embedded in para$n. Fivemicrometer slices were stained with hematoxylin}eosin (HE) and examined by light microscopy. Data are reported as mean$SEM and were analyzed statistically by one-way analysis of variance (ANOVA) followed by Dunnett's test to determine the di!erences between the established study points within groups. The level of signi"cance was set at P(0.05. The statistical analyses were done using GraphPad Instat v.2.01 software (1993). RESULTS
Body weight gain is found in Fig. 1. No signi"cant di!erence was observed in any experimental group compared to controls, at any period of the experiment. Similarly, food consumption did not di!er between experimental groups and controls (data not shown). No deaths or clinical signs of toxicity occurred in any rat throughout the experimental period. No di!erences (P'0.05) were found in plasma glucose, T3, or T4 levels in any group (Table 1). On the other hand, plasma cholesterol levels in the rats treated with 0.6 mg KCN/kg/day were signi"cantly lower than those levels from controls. A comparative study of sections from the pancreas and thyroid glands revealed no lesions in any group. The histopathologic study of the CNS revealed neuronal loss in the hippocampus, which was more intense in animals from the 0.6 mg KCN/kg group. In the spinal cord, spheroids were found on white matter from every experimental group; quantitatively, these spheroids were positively correlated with increasing dose. Furthermore, examination of the cerebellum from animals that received higher cyanide doses revealed damaged Purkinje cells and loss of cerebellar white matter. DISCUSSION
Reduction of weight gain associated with chronic cyanide exposure was found in several animal species, such as rats
39
LONG-TERM EFFECTS OF CYANIDE
TABLE 1 Plasma Levels of Glucose, Cholesterol, Thyroxine (T4), and Triiodothyronine (T3) from Rats That Received Di4erent Doses of Potassium Cyanide (KCN) for 12 Weeks KCN
Glucose (in mg/dL) Cholesterol (in mg/dL) T4 (in g/dL) T3 (in ng/dL)
Control (n"7)
0.15 mg/kg/day (n"6)
0.3 mg/kg/day (n"6)
0.6 mg/kg/day (n"7)
128.1$18.8 35.7$4.18 2.63$0.27 65.8$6.12
136.0$21.3 39.7$2.06 2.60$0.21 61.9$5.98
134.7$01.6 26)6$2.77 2.65$0.32 65.9$7.10
128.9$12.7 19.9$3.05* 2.66$0.30 66.5$6.76
* Signi"cantly di!erent from control group (P(0.05, ANOVA followed by Dunnett's test).
(Philbrick et al., 1979), dogs (Ibebunjo et al., 1992), pigs (Tewe et al., 1984), sheep (Onwuka et al., 1992), and goats (Onwuka et al., 1992; Soto-Blanco et al., 2001), as well as humans (Blanc et al., 1985). However, current results found no di!erences between control and experimental rats in this parameter. The body weight reduction produced by cyanide appears to be a result of sulfur-containing amino acid depletion caused by their use as a source for sulfur for cyanide detoxi"cation (Onwuka et al., 1992). In fact, Philbrick et al. (1979), who also used rats as an animal model to study the chronic e!ects of the cyanogenic glycoside, linamarin, veri"ed a reduced weight gain only in rats from the experimental group whose ration had been depleted of the sulfur amino acid methionine. It has also been proposed that impairment of body development could be a consequence of hypothyroidism induced by chronic cyanide exposure, since reduced T3 levels may promote an impaired secretion of growth hormone and a reduced number of growth hormone receptors (SotoBlanco et al., 2001). In the presented study, the plasma T4 and T3 levels of experimental animals did not di!er from those of the control group, and cholesterol levels were not increased in treated groups. Furthermore, the morphology of the thyroid follicles was una!ected by cyanide administration. Thus, it is reasonable to assume that the lack of a toxic e!ect on the thyroid could also be responsible for the unchanged weight gain observed in the experimental rats in our study. Cyanide has been pointed out as being responsible for the pancreatic diabetes occurring in humans who regularly ingest inadequately detoxi"ed cassava (McMillan and Geevarghese, 1979). However, in the present study no diabetogenic e!ect was found by biochemical or histological approaches, in agreement with results reported by others (Kamalu, 1991; Akanji and Famuyiwa, 1993; Okolie and Osagie, 2000; Soto-Blanco et al., 2001). Furthermore, in the literature there are only three experimental studies (Kamalu, 1991; Geldof et al., 1992; Akanji and Famuyiwa, 1993) that report diabetes as a toxic e!ect produced by
feeding cassava to animals. Thus, it is possible that cyanide itself does not induce pancreatic lesions. In fact, Kamalu (1995) suggested that linamarin, the major cyanogenic glycoside of cassava, and not cyanide, inhibits Na>K>ATPase activity in pancreas, thus producing diabetes. On the other hand, several epidemiological surveys conducted on humans did not detect any relationship between cassava feeding and the development of diabetes (Teuscher et al., 1987; Cooles, 1988; Vannasaeng et al., 1988; Sarles, 1992; Narendranathan and Cheriyan, 1994). Several neuropathies, such as TAN, konzo, and ocular pathologies, have been associated with chronic cyanide exposure (Osuntokun, 1981; Tucker and Hedges, 1993; TylleskaK r et al., 1995; Solberg et al., 1998). However, histopathological examinations of material from these neurological diseases are lacking. On the other hand, some natural cases and experimental reproduction of this exposure in animals are available in the literature. Thus, damage at the CNS level had been described in lambs (Bradley et al., 1995), horses (Adams et al., 1969), and cattle (McKenzie and McMicking, 1977) fed Sorghum. An experimental study carried out on mice subjected to prolonged treatment with KCN found lesions mainly in the substantia nigra and cortex (Mills et al., 1999). Also, literature studies conducted on rats have found that high doses of cyanide administered chronically to these animals produced CNS lesions such as degeneration of neurons in the hippocampus and cortex and of Purkinje cells in the cerebellum (Smith et al., 1963) and vacuolation and gliosis in the spinal cord and white matter (Philbrick et al., 1979). The present study found lesions similar to those described above; however, contrary to these former studies, low doses of cyanide were used here. Thus, in contrast to the statement of Way (1984), who reported that the dosage of cyanide required to produce damage in the CNS of rats is in the lethal range, we veri"ed that small quantities of cyanide administered over a prolonged period of time (12 weeks) is also able to produce neurological lesions in this laboratory animal.
40
SOTO-BLANCO, MARIOKA, AND GOD RNIAK
In conclusion, the present study found that chronic administration of cyanide to rats could promote neuropathological lesions without a!ecting pancreas or thyroid gland metabolism. Thus, it could be suggested that the rat is an excellent animal model to assess the chronic toxicity of cyanide to the CNS. On the other hand, it is probably a poor model to evaluate its goitrogenic and diabetogenic e!ects; however, it should be considered that there are controversies about this late consequence attributable to cyanide. Thus, it is "rst necessary to verify whether, in fact, cyanide has toxic e!ects at the pancreas level. REFERENCES Adams, L. G., Dollahite, J. W., Romane, W. M., Bullard, T. L., and Bridges, C. H. (1969). Cystitis and ataxia associated with sorghum ingestion by horses. J. Am.
Kamalu, B. P. (1991). The e!ect of a nutritionally-balanced cassava (Manihot esculenta Crantz) diet on endocrine function using the dog as a model. 1. Pancreas. Br. J. Nutr. 65, 365}372. Kamalu, B. P. (1995). The adverse e!ects of long-term cassava (Manihot esculenta Crantz) consumption. Int. J. Food Sci. Nutr. 46, 65}93. McKenzie, R. A., and McMicking, L. I. (1977). Ataxia and urinary incontinence in cattle grazing sorghum. Aust.
Bradley, G. A., Metcalf, H. C., Reggiardo, C., Noon, T. H., Bicknell, E. J., Lozano-Alarcon, F., Reed, R. E., and Riggs, M. W. (1995). Neuroaxonal degeneration in sheep grazing sorghum pastures. J.
Pitchumoni, C. S., Jain, N. K., Lowenfels, A. B., and DiMagno, E. P. (1988). Chronic cyanide poisoning: Unifying concept for alcoholic and tropical pancreatitis. Pancreas 3, 220}222.
Cooles, P. (1988). Diabetes and cassava in Dominica. ¹rop. Geog. Med. 40, 272}273.
Poulton, J. E. (1983). Cyanogenic compounds in plants and their toxic e!ects. In Handbook of Natural ¹oxins*Plant and Fungal toxins (R. F. Keeler and A. T. Tu, Eds.), pp. 117}157. Dekker, New York.
Dohan, O., De la Vieja, A., and Carrasco, N. (2000). Molecular study of the sodium-iodide symporter (NIS): A new "eld in thyroidology. ¹rends Endocrinol. Metab. 11, 99}105.
Ratnakumar, J. N., and Rajan, A. (1992). Goitrogenic e!ect of cassava in broiler rabbits. Ind. J. Anim. Sci. 62, 670}676.
Freeman, A. G. (1988). Optic neuropathy and chronic cyanide intoxication: A review. J. R. Soc. Med. 81, 103}106. Gaitan, E., Cooksey, R. C., Legan, J., Lindsay, R. H., Ingbar, S. H., and Medeiros-Neto, G. (1994). Antithyroid e!ects in vivo and in vitro of babassu and mandioca: A staple food in goitre areas of Brazil. Eur. J. Endocrinol. 131, 138}144. Geldof, A. A., Becking, J. L., and de Vries, C. D. (1992). Histopathological changes in rat pancreas after fasting and cassava feeding. In
Sarles, H. (1992). Chronic pancreatitis and diabetes. Bailliers Clin. Endocrinol. Metab. 6, 745}775. Schulz, V. (1984). Clinical pharmacokinetics of nitroprusside, cyanide, thiosulphate and thiocyanate. Clin. Pharmacokinet. 9, 239}251. Smith, A. D. M., Duckett, S., and Waters, A. H. (1963). Neuropathological changes in chronic cyanide intoxication. Nature 200, 179}181. Solberg, Y., Rosner, M., and Belkin, M. (1998). The association between cigarette smoking and ocular diseases. Surv. Ophthalmol. 42, 535}547. Soto-Blanco, B., GoH rniak, S. L., and Kimura, E. T. (2001). Physiopathological e!ects of the administration of chronic cyanide to growing goats* A model for ingestion of cyanogenic plants.
LONG-TERM EFFECTS OF CYANIDE Tsao, K., Aikene, P. A., and Johns, D. R. (1999). Smoking as an aetiological factor in a pedigree with Leber's hereditary optic neuropathy. Br. J. Ophthalmol. 83, 577}581. Tucker, K., and Hedges, T. R. (1993). Food shortages and an epidemic of optic and peripheral neuropathy in Cuba. Nutr. Rev. 51, 349}357. TylleskaK r, T., Banea, M., Bikangi, N., Nahimana, G., Persson, L. As ., and Rosling, H. (1995). Dietary determinants of a non-progressive spastic
41
paraparesis (Konzo): A case}referent study in a high incidence area of Zaire. Int. J. Epidemiol. 24, 949}956. Vannasaeng, S., Nitiyanant, W., and Vichayanrat, A. (1988). Case}control study on risk factors associated with "brocalculous pancreatic diabetes. Diabetes Med. 5, 835}939. Way, J. L. (1984). Cyanide intoxication and its mechanism of antagonism. Annu. Rev. Pharmacol. ¹oxicol. 24, 451}481.
Effects of Long-Term Low-Dose Cyanide Administration to Rats1 Benito Soto-Blanco, Paulo CeH sar Marioka, and Silvana Lima GoH rniak Research Center for Veterinary Toxicology, Department of Pathology, School of Veterinary Medicine, University of Sa o Paulo, Brazil Received October 25, 2000
feeding have cyanogenic glycosides that release cyanide. The most important cyanogenic plant consumed by humans and animals worldwide is cassava (Manihot esculenta Crantz), which contains mainly linamarin as its cyanogenic glycoside (Poulton, 1983). With respect to the mechanism of action, inhibition of cellular respiration is produced by binding of cyanide to ferric iron in cytochrome oxidase, resulting in histotoxic cellular hypoxia or anoxia. Nevertheless, mammals have an e$cient mechanism of detoxi"cation of this poison through conversion to thiocyanate by rhodanase, a mitochondrial enzyme (Way, 1984). Acute toxicity has been extensively studied, but there is a paucity of information about chronic cyanide exposure. On the other hand, it has been assumed that the more widely distributed adverse e!ects of cyanide are due its chronic toxicity. Probably the most pathologic conditions attributable to prolonged exposure to cyanide are the neuropathies. Thus, degenerative diseases such as tropical ataxic neuropathy (TAN) (Osuntokun, 1981) and spastic paraparesis or &&konzo'' (TylleskaK r et al., 1995) have been associated with high cassava consumption. Furthermore, prolonged cyanide exposure through both tobacco smoke and cassava consumption may be implicated in the pathogenesis of ocular pathologies such as tobacco}alcohol amblyopia (Solberg et al., 1998), retrobulbar neuropathy of pernicious anemia (Freeman, 1988), Leber's hereditary optic neuropathy (Tsao et al., 1999), and Cuban optic neuropathy (Tucker and Hedges, 1993). Horses, cattle, and sheep grazing on cyanogenic Sorghum pastures developed neuronal disorders, especially ataxia (Adams et al., 1969; McKenzie and McMicking, 1977; Bradley et al., 1995). There are many studies reporting a correlation between cyanogenic plant ingestion and development of goiter in di!erent animal species (Ratnakumar and Rajan, 1992; Tewe et al., 1984; Gutzwiller, 1993) and humans (Gaitan et al., 1994). Goiter is related to the main product of transformation of cyanide, thiocyanate, which competes with iodide in the Na>/l\ symporter in the thyroid gland, consequently inhibiting the synthesis and clearance of thyroid hormones (Dohan et al., 2000).
Chronic exposure to cyanide has been associated with development of pancreatic diabetes, hypothyroidism, and several neurological diseases in both humans and animals. However, there is a limited number of experimental models for these pathologies. Thus, in the present study 0, 0.15, 0.3, or 0.6 mg KCN/kg body weight/day was administered for 3 months to 26 rats. On the last day, plasma samples were obtained for glucose, cholesterol, and thyroidal hormone measurement, and the pancreas, thyroids, and whole central nervous system were collected for histopathologic study. There were no signi5cant di4erence in plasma concentrations of the substances measured between groups, and no lesions were found in the pancrease or thyroid. The CNS of experimental animals revealed the presence of spheroids on the ventral horn of the spinal cord, neuron loss in the hippocampus, damaged Purkinje cells, and loss of cerebellar white matter. In conclusion, cyanide administration could promote neuropathological lesions in rats without a4ecting pancreas or thyroid gland metabolism. 2002 Elsevier Science (USA) Key Words: cyanide; tropical diabetes; hypothyroidism; goiter; neurotoxicity.
INTRODUCTION
Cyanide is a ubiquitous toxic ion in the environment. Exposure to this substance occurs through several sources. Cyanide is present in many industrial processes such as hardening of metals, electroplating, chemical synthesis, and gold extraction. Environmental pollution can be produced by waste discharges from these industries (Poulton, 1983; Boening and Chew, 1999). This ion is also found in smoke from tobacco, "res, and propulsion motors (Poulton, 1983). Some drugs, such as nitroprusside and laetrile, can release cyanide (Moertel et al., 1981; Schulz, 1984). Furthermore, a large number of plant species for human and animal This study was supported by a grant from FAPESP (Fundac,a o de Amparo a` Pesquisa do Estado de Sa o Paulo). To whom correspondence should be addressed at Research Center for Veterinary Toxicology (CEPTOX), Department of Pathology, School of Veterinary Medicine, University of Sa o Paulo, Av. Orlando Marques de Paiva 87, Sa o Paulo 05508-900, Brazil. E-mail: [email protected]. 37
0147-6513/02 $35.00 2002 Elsevier Science (USA) All rights reserved.
38
SOTO-BLANCO, MARIOKA, AND GOD RNIAK
Furthermore, the development of pancreatic diabetes, known as tropical diabetes, in humans has been associated with consumption of cyanogenic plants, especially cassava (McMillan and Geevarghese, 1979; Assan et al., 1988; Pitchumoni et al., 1988; Kamalu, 1995). McMillan and Geevarghese (1979) suggested that pancreatic diabetes could be produced by the action of cyanide release by cassava on the acinar portion of the pancreas, thus a!ecting the endocrine portion. However, no experimental model reproducing pancreatic diabetes by administration of cyanide itself is available in the literature. In spite of the widespread distribution of cyanide-containing sources and its possible implication as the etiology of various human and animal diseases, it has been di$cult to precisely assess the contribution of this substance to the clinical disease, since most of the evidence is based on "eld studies without controlled experimental conditions. Thus, the purpose of the present study was to determine the e!ects of long-term low-dose potassium cyanide (KCN) administration on the central nervous system (CNS) and thyroid and pancreatic metabolism, using rats as an animal model. MATERIAL AND METHODS
Used were 26 weanling male Wistar rats, 22 days old, weighing on average 44 g at the beginning of the experiment. The animals were kept in groups of 3 or 4 in 33;41-cm acrylic cages at 22}243C on a 10-h light} 14-h dark cycle (lights on at 7:00 AM) with free access to food (Nutrilab, Brazil) and water. The animals were randomly divided into four groups, dosed with 0 (7 animals), 0.15 (6 animals), 0.3 (6 animals), or 0.6 (7 animals) mg KCN/body weight/day by gavage every day for 3 months. These KCN doses are 1.5, 3.0, and 6.0% of the peroral LD for male rats (10 mg/kg) (Hayes, 1967). Cyanide was always administered at the same time of day (9:00 to 10:00 AM), and KCN solutions were prepared fresh every day. The health of the rats was checked daily, body weight was recorded twice a week to adjust the KCN dose, and food consumption was recorded on the same day as body weight. At the end of the experiment, blood samples were collected from the hepatic vein of rats under ether anesthesia with heparinized (Liquemine, Roche, Brazil) syringes (SR, Brazil) in order to obtain plasma. These samples were frozen at !103C until analysis. Plasma cholesterol and glucose levels were determined using a commercial kit (Laborlab, Brazil), and thyroxine (T4) and triiodothyronine (T3) levels were measured by radioimmunoassay (DPC). After blood collection, the animals were killed by transcardiac perfusion with 10% bu!ered formalin followed by Carnoy solution. Whole CNS, pancreas, and thyroid glands were collected for histopathologic study. After complete "xation, the CNS was cut into fragments containing cortex, hippocampus, brain stem (caudal midbrain, pons and
FIG. 1. Body weight gain of rats treated with di!erent KCN doses.
medulla oblongata), cerebellum, and spinal cord. CNS and thyroid gland fragments were embedded in para$n. Fivemicrometer slices were stained with hematoxylin}eosin (HE) and examined by light microscopy. Data are reported as mean$SEM and were analyzed statistically by one-way analysis of variance (ANOVA) followed by Dunnett's test to determine the di!erences between the established study points within groups. The level of signi"cance was set at P(0.05. The statistical analyses were done using GraphPad Instat v.2.01 software (1993). RESULTS
Body weight gain is found in Fig. 1. No signi"cant di!erence was observed in any experimental group compared to controls, at any period of the experiment. Similarly, food consumption did not di!er between experimental groups and controls (data not shown). No deaths or clinical signs of toxicity occurred in any rat throughout the experimental period. No di!erences (P'0.05) were found in plasma glucose, T3, or T4 levels in any group (Table 1). On the other hand, plasma cholesterol levels in the rats treated with 0.6 mg KCN/kg/day were signi"cantly lower than those levels from controls. A comparative study of sections from the pancreas and thyroid glands revealed no lesions in any group. The histopathologic study of the CNS revealed neuronal loss in the hippocampus, which was more intense in animals from the 0.6 mg KCN/kg group. In the spinal cord, spheroids were found on white matter from every experimental group; quantitatively, these spheroids were positively correlated with increasing dose. Furthermore, examination of the cerebellum from animals that received higher cyanide doses revealed damaged Purkinje cells and loss of cerebellar white matter. DISCUSSION
Reduction of weight gain associated with chronic cyanide exposure was found in several animal species, such as rats
39
LONG-TERM EFFECTS OF CYANIDE
TABLE 1 Plasma Levels of Glucose, Cholesterol, Thyroxine (T4), and Triiodothyronine (T3) from Rats That Received Di4erent Doses of Potassium Cyanide (KCN) for 12 Weeks KCN
Glucose (in mg/dL) Cholesterol (in mg/dL) T4 (in g/dL) T3 (in ng/dL)
Control (n"7)
0.15 mg/kg/day (n"6)
0.3 mg/kg/day (n"6)
0.6 mg/kg/day (n"7)
128.1$18.8 35.7$4.18 2.63$0.27 65.8$6.12
136.0$21.3 39.7$2.06 2.60$0.21 61.9$5.98
134.7$01.6 26)6$2.77 2.65$0.32 65.9$7.10
128.9$12.7 19.9$3.05* 2.66$0.30 66.5$6.76
* Signi"cantly di!erent from control group (P(0.05, ANOVA followed by Dunnett's test).
(Philbrick et al., 1979), dogs (Ibebunjo et al., 1992), pigs (Tewe et al., 1984), sheep (Onwuka et al., 1992), and goats (Onwuka et al., 1992; Soto-Blanco et al., 2001), as well as humans (Blanc et al., 1985). However, current results found no di!erences between control and experimental rats in this parameter. The body weight reduction produced by cyanide appears to be a result of sulfur-containing amino acid depletion caused by their use as a source for sulfur for cyanide detoxi"cation (Onwuka et al., 1992). In fact, Philbrick et al. (1979), who also used rats as an animal model to study the chronic e!ects of the cyanogenic glycoside, linamarin, veri"ed a reduced weight gain only in rats from the experimental group whose ration had been depleted of the sulfur amino acid methionine. It has also been proposed that impairment of body development could be a consequence of hypothyroidism induced by chronic cyanide exposure, since reduced T3 levels may promote an impaired secretion of growth hormone and a reduced number of growth hormone receptors (SotoBlanco et al., 2001). In the presented study, the plasma T4 and T3 levels of experimental animals did not di!er from those of the control group, and cholesterol levels were not increased in treated groups. Furthermore, the morphology of the thyroid follicles was una!ected by cyanide administration. Thus, it is reasonable to assume that the lack of a toxic e!ect on the thyroid could also be responsible for the unchanged weight gain observed in the experimental rats in our study. Cyanide has been pointed out as being responsible for the pancreatic diabetes occurring in humans who regularly ingest inadequately detoxi"ed cassava (McMillan and Geevarghese, 1979). However, in the present study no diabetogenic e!ect was found by biochemical or histological approaches, in agreement with results reported by others (Kamalu, 1991; Akanji and Famuyiwa, 1993; Okolie and Osagie, 2000; Soto-Blanco et al., 2001). Furthermore, in the literature there are only three experimental studies (Kamalu, 1991; Geldof et al., 1992; Akanji and Famuyiwa, 1993) that report diabetes as a toxic e!ect produced by
feeding cassava to animals. Thus, it is possible that cyanide itself does not induce pancreatic lesions. In fact, Kamalu (1995) suggested that linamarin, the major cyanogenic glycoside of cassava, and not cyanide, inhibits Na>K>ATPase activity in pancreas, thus producing diabetes. On the other hand, several epidemiological surveys conducted on humans did not detect any relationship between cassava feeding and the development of diabetes (Teuscher et al., 1987; Cooles, 1988; Vannasaeng et al., 1988; Sarles, 1992; Narendranathan and Cheriyan, 1994). Several neuropathies, such as TAN, konzo, and ocular pathologies, have been associated with chronic cyanide exposure (Osuntokun, 1981; Tucker and Hedges, 1993; TylleskaK r et al., 1995; Solberg et al., 1998). However, histopathological examinations of material from these neurological diseases are lacking. On the other hand, some natural cases and experimental reproduction of this exposure in animals are available in the literature. Thus, damage at the CNS level had been described in lambs (Bradley et al., 1995), horses (Adams et al., 1969), and cattle (McKenzie and McMicking, 1977) fed Sorghum. An experimental study carried out on mice subjected to prolonged treatment with KCN found lesions mainly in the substantia nigra and cortex (Mills et al., 1999). Also, literature studies conducted on rats have found that high doses of cyanide administered chronically to these animals produced CNS lesions such as degeneration of neurons in the hippocampus and cortex and of Purkinje cells in the cerebellum (Smith et al., 1963) and vacuolation and gliosis in the spinal cord and white matter (Philbrick et al., 1979). The present study found lesions similar to those described above; however, contrary to these former studies, low doses of cyanide were used here. Thus, in contrast to the statement of Way (1984), who reported that the dosage of cyanide required to produce damage in the CNS of rats is in the lethal range, we veri"ed that small quantities of cyanide administered over a prolonged period of time (12 weeks) is also able to produce neurological lesions in this laboratory animal.
40
SOTO-BLANCO, MARIOKA, AND GOD RNIAK
In conclusion, the present study found that chronic administration of cyanide to rats could promote neuropathological lesions without a!ecting pancreas or thyroid gland metabolism. Thus, it could be suggested that the rat is an excellent animal model to assess the chronic toxicity of cyanide to the CNS. On the other hand, it is probably a poor model to evaluate its goitrogenic and diabetogenic e!ects; however, it should be considered that there are controversies about this late consequence attributable to cyanide. Thus, it is "rst necessary to verify whether, in fact, cyanide has toxic e!ects at the pancreas level. REFERENCES Adams, L. G., Dollahite, J. W., Romane, W. M., Bullard, T. L., and Bridges, C. H. (1969). Cystitis and ataxia associated with sorghum ingestion by horses. J. Am.
Kamalu, B. P. (1991). The e!ect of a nutritionally-balanced cassava (Manihot esculenta Crantz) diet on endocrine function using the dog as a model. 1. Pancreas. Br. J. Nutr. 65, 365}372. Kamalu, B. P. (1995). The adverse e!ects of long-term cassava (Manihot esculenta Crantz) consumption. Int. J. Food Sci. Nutr. 46, 65}93. McKenzie, R. A., and McMicking, L. I. (1977). Ataxia and urinary incontinence in cattle grazing sorghum. Aust.
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Pitchumoni, C. S., Jain, N. K., Lowenfels, A. B., and DiMagno, E. P. (1988). Chronic cyanide poisoning: Unifying concept for alcoholic and tropical pancreatitis. Pancreas 3, 220}222.
Cooles, P. (1988). Diabetes and cassava in Dominica. ¹rop. Geog. Med. 40, 272}273.
Poulton, J. E. (1983). Cyanogenic compounds in plants and their toxic e!ects. In Handbook of Natural ¹oxins*Plant and Fungal toxins (R. F. Keeler and A. T. Tu, Eds.), pp. 117}157. Dekker, New York.
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Freeman, A. G. (1988). Optic neuropathy and chronic cyanide intoxication: A review. J. R. Soc. Med. 81, 103}106. Gaitan, E., Cooksey, R. C., Legan, J., Lindsay, R. H., Ingbar, S. H., and Medeiros-Neto, G. (1994). Antithyroid e!ects in vivo and in vitro of babassu and mandioca: A staple food in goitre areas of Brazil. Eur. J. Endocrinol. 131, 138}144. Geldof, A. A., Becking, J. L., and de Vries, C. D. (1992). Histopathological changes in rat pancreas after fasting and cassava feeding. In
Sarles, H. (1992). Chronic pancreatitis and diabetes. Bailliers Clin. Endocrinol. Metab. 6, 745}775. Schulz, V. (1984). Clinical pharmacokinetics of nitroprusside, cyanide, thiosulphate and thiocyanate. Clin. Pharmacokinet. 9, 239}251. Smith, A. D. M., Duckett, S., and Waters, A. H. (1963). Neuropathological changes in chronic cyanide intoxication. Nature 200, 179}181. Solberg, Y., Rosner, M., and Belkin, M. (1998). The association between cigarette smoking and ocular diseases. Surv. Ophthalmol. 42, 535}547. Soto-Blanco, B., GoH rniak, S. L., and Kimura, E. T. (2001). Physiopathological e!ects of the administration of chronic cyanide to growing goats* A model for ingestion of cyanogenic plants.
LONG-TERM EFFECTS OF CYANIDE Tsao, K., Aikene, P. A., and Johns, D. R. (1999). Smoking as an aetiological factor in a pedigree with Leber's hereditary optic neuropathy. Br. J. Ophthalmol. 83, 577}581. Tucker, K., and Hedges, T. R. (1993). Food shortages and an epidemic of optic and peripheral neuropathy in Cuba. Nutr. Rev. 51, 349}357. TylleskaK r, T., Banea, M., Bikangi, N., Nahimana, G., Persson, L. As ., and Rosling, H. (1995). Dietary determinants of a non-progressive spastic
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paraparesis (Konzo): A case}referent study in a high incidence area of Zaire. Int. J. Epidemiol. 24, 949}956. Vannasaeng, S., Nitiyanant, W., and Vichayanrat, A. (1988). Case}control study on risk factors associated with "brocalculous pancreatic diabetes. Diabetes Med. 5, 835}939. Way, J. L. (1984). Cyanide intoxication and its mechanism of antagonism. Annu. Rev. Pharmacol. ¹oxicol. 24, 451}481.