Veterinary Research Communications, 31 (2007) 93104 DOI: 10.

1007/s11259-006-3361-x

Springer 2007

Effects of Long-term Cyanide Ingestion by Pigs

H. Manzano1 , A. Benedito de Sousa1 , B. Soto-Blanco2 , J.L. Guerra3 , P.C. Maiorka3 and S.L. G orniak1, 1 Research Center for Veterinary Toxicology (CEPTOX), Department of Pathology, School o of Veterinary Medicine, S ao Paulo, Brazil; 2 Department of Veterinary Medicine, Mossor Agriculture School, Mossor o, Brazil; 3 Department of Pathology, School of Veterinary Medicine, S ao Paulo, Brazil Correspondence: E-mail: gorniak@usp.br

ABSTRACT
Animal performance and health status are adversely affected by long-term cyanide ingestion; however, the effects of cyanide ingestion by pigs have not been fully determined. The aim of the present study was to determine the effects of prolonged exposure to different doses of potassium cyanide (KCN) in growing-nishing swine. Twenty-four pigs, 45 days of age, were divided into four equal groups and treated with different doses of KCN: 0, 2.0, 4.0 or 6.0 mg per kg body weight per day for 70 consecutive days. The results showed a signicant alteration in thiocyanate, creatinine and urea levels and in alanine aminotransferase activity of swine dosed with 4.0 and 6.0 mg/kg/KCN. Thyroid weight was signicantly increased in those pigs from 4.0 mg/kg KCN group, but no change in cholesterol, triiodothyronine or thyroline levels were observed. Body and carcase weights, body weight gain, and bacon thickness were not affected by KCN treatment. The histopathological study revealed increased numbers of vacuoles in the colloid of thyroid follicles, degeneration of cerebellar white matter and Purkinje cells, degeneration of renal tubular epithelial cells, caryolysis and pyknosis in hepatocytes, and disturbance of the normal lobular architecture of the liver in all treated pigs. Thus, long-term administration of KCN to swine affects several tissues and could adversely affect animal production. Keywords: cyanide, pigs, chronic toxicity, cassava Abbreviations: KCN, potassium cyanide; ALT, alanine aminotransferase; BUN, blood urea nitrogen; T4 , thyroxine; T3 , triiodothyronine; HE, haematoxylineosin; CNS, central nervous system; SAA, sulphur-containing amino acid

INTRODUCTION Pigs require large quantities of carbohydrates (energy) and moderate quantities of protein for growth and development. In the leading pork-production areas, large supplies of grains available at a reasonable price provide this energy source. In other countries, production of cereal grain is insufcient even for human consumption needs. However, these countries have the potential to produce or are already producing large quantities of other feed sources that, if properly fed and supplemented, could support a large and efcient swine industry. One feed source with a high and still unexploited potential in many tropical regions is Manihot esculenta Cranes, commonly known as cassava, yucca, manioc, tapioca, or mandioca. In fact, cassava is an extensively cultivated plant in tropical countries, where its starchy root tubers represent a major source of dietary carbohydrate for humans and livestock (Philbrick et al., 1977). It has been estimated that about 65% of the cassava crop is used for human consumption, while the remainder is used to feed animals, including swine (Oke, 1984), to produce starch, and for industrial applications.

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While cassava production is of low cost, permitting its widespread use as a source of carbohydrates for animal feeding, with the tuber being extensively used as a corn substitute to supply energy in animal feed, it is well known that cassava contains cyanogenic glycosides, known as linamarin and lotaustralin, that could cause toxicity. In fact, long-term ingestion of low amounts of cyanide by humans has been linked to several neuropathies and to goitre (Osuntokun, 1981; Way, 1984; Rosling, 1994). Furthermore, experimental studies on different animal species have shown that chronic cyanide ingestion causes impaired body growth, goitre and lesions of the central nervous system (Kamalu, 1995; Soto-Blanco et al., 2001; Sousa et al., 2002). Cyanide has also been suggested to be responsible for tropical diabetes in humans (McMillan and Geevarghese, 1979). It has been assumed that the more widely distributed adverse effects of cyanide are due to chronic toxicity, so that there is a need to carefully assess the effect of prolonged exposure to cyanide. Although several studies on chronic cyanide intoxication have been conducted in various animal species, very little is known about its effects on swine, and data obtained for other species should not be directly extrapolated to pigs because of differences in cyanide pharmacokinetics (Sousa et al., 2003). Thus, the objective of the present study was to determine the effect of prolonged exposure to potassium cyanide (KCN) in growingnishing swine.

MATERIAL AND METHODS Animals and experimental design Twenty-four Landrace-Large White pigs, 45 days old, were randomly divided into four equal groups, which were dosed with 0 (control), 2.0, 4.0, and 6.0 mg of KCN per kg body weight per day for 70 consecutive days. KCN was administered twice a day, with half the dose being given between 07:30 and 08:00 and the other half between 15:30 and 16:00, mixed with 10 g of starch biscuits in individual troughs. The control group received only the biscuits. All animals were weighed weekly and the body weight gains were computed. Blood samples (10 ml) were collected from the external carotid vein, using heparinized (Liquemine, Roche, Rio de Janeiro, Brazil) syringes (SR, S ao Paulo, Brazil), before the experimental period and then every other week. Blood samples were held at 10 C until analysis. Alanine aminotransferase (ALT) activity and glucose, cholesterol, blood urea nitrogen (BUN) and creatinine concentrations were determined colorimetrically in serum using commercial kits (Merck, Darmstad, Germany). Thyroid hormone levels, thyroxine (T4 ) and triiodothyronine (T3 ) were measured in serum by radioimmunoassay using Count a Count kits from DPC (Los Angeles, CA, USA). Thiocyanate levels in plasma of swine were estimated by ion-exchange resin IRA 96 (Sigma, St Louis, MO, USA), and measured by colorimetric assay with a spectrophotometer using a methodology proposed by Tominaga and Midio (1991) as modied by Siqueira and colleages (1996). This method was adapted in our laboratory to measure the thiocyanate in 1.0 ml of swine plasma. All the reagents used in the assay were of reagent grade and solutions were prepared with deionized water.

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At the end of the experimental period, all the animals were killed, thyroid glands and carcases were weighed, and bacon thickness was measured from the 10th and 11th thoracic vertebrae. Carcase efciency was calculated by the ratio of carcase weight to live weight. Fragments of whole CNS, thyroid glands, pancreas, liver and kidneys were collected, xed, and stored in 10% buffered formalin for histological examination. The fragments were embedded in parafn blocks and 5 m sections were stained with haematoxylin and eosin (HE). Statistical analysis Data are reported as means SEM and were analysed statistically by one-way analysis of variance (ANOVA) followed by Dunnetts test, with the level of signicance set at p < 0.05. The statistical analyses were processed using the GraphPad Instat, 1993, v. 2.01 software.

RESULTS Clinical observations revealed no alteration in any animal evaluated, except for one pig from the 3.0 mg KCN/kg per day group, which showed slavering and breathing difculty immediately after KCN administration on the 35th day of the experiment. No signicant differences were found in body weight gain, carcase weight, carcase efciency or bacon thickness between the treated and control groups. However, an increase ( p < 0.05) in the weight of the thyroid glands from pigs treated with 6.0 mg/kg KCN was observed compared to the control group (Table I). Plasma thiocyanate levels were increased from the 2nd and 7th weeks of the experiment in pigs treated with 6.0 and 4.0 mg/kg KCN, respectively. ALT activities from pigs treated with the highest doses of KCN were signicantly increased ( p < 0.05) from the 42nd day of the experiment to the end of the trial; but animals that received 2.0 mg/kg of KCN showed an increase in this enzyme only on the last day of cyanide administration (Table II).
TABLE I Body weight gain, carcase weight and efciency and weight of thyroid gland in growingnishing pigs that received different doses of KCN with 10 g of the biscuits KCN (mg/kg per day)a Parameter Body weight gain (kg) Carcase weight (kg) Carcase efciency (%) Weight of thyroid gland 0.0 47.9 9.1 50.6 14.4 74.0 3.6 5.8 0.9 2.0 46.3 8.2 53.9 9.9 78.7 7.5 6.0 0.8 4.0 48.2 10.7 53.3 12.1 78.3 6.5 5.7 1.1 6.0 51.4 10.6 56.3 8.5 76.5 2.7 7.2 1.5

a n = 10 for each group (mean SEM) p < 0.05 compared to control (ANOVA

followed by Dunnett test)

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TABLE II Plasma concentrations of thiocyanate (mol/L) in growing-nishing pigs that received different doses of potassium cyanide (KCN)a KCN (mg/kg per day)a Day of experiment 0 14 28 42 56 70 0.0 41.1 4.4(10)b 46.2 12.7(10) 36.6 7.1(9) 31.3 17.2(10) 25.1 13.7(10) 30.3 8.0(10) 2.0 47.0 3.1(6) 55.4 16.2(10) 37.1 10.3(9) 41.0 6.3(10) 46.0 16.5(10) 47.8 6.3(10) 4.0 45.2 6.8(8) 56.4 15.9(10) 42.1 11.3(10) 57.7 15.0 (9) 70.2 24.3 (10) 63.5 18.5 (10) 6.0 43.2 6.6(10) 67.9 10.6 (10) 52.1 19.8 (9) 54.5 27.8 (10) 76.0 28.9 (10) 70.1 29.6 (10)

a Values are mean SEM b Number of animals used is given in parentheses p < 0.05 compared to control (ANOVA followed

by Dunnett test)

Creatinine levels uctuated during the trial. Serum BUN concentration was signicantly increased ( p < 0.05) in animals treated with 4.0 and 6.0 mg/kg of KCN from the 4th week of the trial (Table III). On the other hand, no signicant variation was found in glucose or cholesterol levels. In the same manner, T3 and T4 levels did not show signicant differences between the dosed and control groups. The histopathological study revealed alterations in the thyroid glands of all dosed animals, characterized by numerous vacuoles in the colloid of thyroid follicles (Figure 1). The liver of the experimental pigs showed caryolysis and pyknosis in hepatocytes (Figure 2A), and distortion of the normal lobular architecture of the liver (Figure 2B). Kidney tissue presented degeneration of the renal tubular epithelial cells (Figure 3). Sections of the central nervous system revealed minimal degeneration of Purkinje cells (Figure 4) and attenuation of cerebellar white matter in pigs from all groups treated with KCN (Figure 5). All the observed lesions occurred in a doseresponse fashion. In contrast, the pancreas was unaffected by the cyanide treatment. There were no histopathological lesions in tissues from the control group. DISCUSSION For economic reasons, cassava has increasingly been used to replace corn in feeding livestock. As a consequence, the animals are exposed to low but constant amounts of cyanide for prolonged periods (Tewe, 1992). The doses of KCN given in this study were related to the levels of cyanide exposure when swine are fed naturally with cassava (Oke, 1984; Bertol and De Lima, 1999; Yeoh and Sun, 2001). Plasma thiocyanate concentration is a reasonable measure of cyanide exposure, since about 80% of the cyanide absorbed by an individual is rapidly converted to thiocyanate by the enzyme rhodanase (Ansell and Lewis, 1970). Since in the present study plasma

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TABLE III Serum concentrations of ALT, urea and creatinine of pigs treated during 4 months with different doses of potassium cyanide (KCN)a KCN (mg/kg per day) Week 1 0 2.0 4.0 6.0 Week 2 0 2.0 4.0 6.0 Week 4 0 2.0 4.0 6.0 Week 6 0 2.0 4.0 6.0 Week 8 0 2.0 4.0 6.0 Week 10 0 2.0 4.0 6.0
a Values are mean SEM b Number of animals used is p

ALT (U/L)

Urea (mg/dl)

Creatinine (mg/dl)

26.5 4.1 (10)b 29.3 4.2 (10) 27.3 3.2 (10) 25.6 3.5 (10) 24.6 3.3 (5) 16.3 2.9 (5) 28.4 3.3 (5) 25.7 3.9 (5) 21.0 2.5 (5) 21.7 3.7 (5) 21.2 3.4 (5) 22.6 1.9 (5) 19.0 4.8 (5) 24.0 4.8 (5) 28.3 5.6 (5) 28.1 3.7 (5) 32.9 5.9 (5) 29.3 7.1 (5) 17.0 2.7 (5) 17.2 3.2 (5) 31.5 2.2 (10) 23.0 8.5 (10) 20.6 6.8 (10) 22.7 9.5 (10)

26.5 7.7 (5) 22.6 6.2 (5) 29.5 5.0 (5) 26.9 4.5 (5) 24.7 4.5 (5) 26.0 8.1(5) 39.1 7.3 (5) 33.0 5.1 (5) 23.9 2.6 (5) 27.3 1.1(5) 33.9 4.5 (5) 38.2 10.2 (5) 30.4 2.8 (4) 38.5 3.7(4) 42.3 3.7 (4) 40.2 2.5 (4) 30.0 2.8(5) 29.1 3.2(5) 35.6 2.9 (5) 36.0 5.8 (5) 26.0 3.0 (5) 29.1 2.4 (5) 37.3 0.9 (5) 36.5 2.8 (5)

0.96 0.2 (5) 0.64 0.1 (4) 1.19 0.4 (5) 0.94 0.07 (5) 0.87 0.08(5) 1.26 0.1 (5) 1.04 0.1 (5) 1.01 0.2(5) 0.93 0.1(5) 1.02 0.04 (5) 1.01 0.07 (5) 0.92 0.07(5) 0.91 0.1 (5) 1.27 0.2 (5) 1.15 0.05 (5) 1.10 0.07 (5) 1.06 0.1 (5) 1.23 0.1 (5) 1.15 0.04 (5) 1.14 0.06 (5) 0.97 0.1 (5) 1.10 0.07 (5) 1.13 0.03 (5) 1.17 0.06 (5)

given in parentheses < 0.05 compared to control (ANOVA followed by Dunnett test)

thiocyanate levels of pigs from the groups treated with 6 and 4 mg/kg KCN increased signicantly from the 2nd and 4th weeks, respectively, we may assume that animals from both groups were signicantly exposed to cyanide. Reduction of weight gain in chronic cyanide exposure has been observed in several animal species, including pigs (Tewe et al., 1977, 1984). It was proposed that the body weight reduction produced by cyanide is a result of depletion of sulphur-containing amino

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Figure 1. Thyroid follicles from a pig that received 6.0 mg/kg KCN, showing the presence of reabsorption vacuoles in colloid. (HE 25)

acids (SAAs) caused by their use as a source of sulphur for cyanide detoxication (Onwuka, 1992). It is well known that cassava is poor in all amino acids, especially the SAAs, and the replacement of maize with cassava causes imbalance of SAA that leads to a lower efciency of feed conversion (Oke, 1984). However, in the present study, no differences in weight gain, carcase weight or carcase efciency were observed. A possible explanation for the difference between the ndings from Tewes experiments (Tewe et al., 1977, 1984) and ours is that in those studies cassava was used as a source of cyanide, whereas KCN was added to a balanced diet in the present study. As a consequence, the availability of SAA in the diet of the pigs when using cassava was low, resulting in decreased performance. This theory is supported by the study conducted on rats by Philbrick and colleagues (1979), who veried reduced weight gain only in animals from the group that were treated with cyanide but whose ration had been depleted of the SAA methionine. It is well known that thiocyanate, the main cyanide metabolite, competes with iodide in the Na+ /I symporter in the thyroid gland, thus inhibiting the synthesis and clearance of thyroid hormones (Dohan et al., 2000). Reduced T3 levels may cause impaired secretion of growth hormone (Loyd et al., 1990) and a reduced number of growth hormone receptors (Koenig et al., 1987), leading to reduced development. Furthermore, it has also been suggested that impairment of body development could be a consequence of hypothyroidism induced by chronic cyanide exposure (Kamalu and Aghranya, 1991; Rosling, 1994). In the present investigation, although the histopathological study revealed increases in the number of vacuoles in the follicular colloid of the thyroid gland from all experimental pigs and in the weight of the thyroid gland from the animals treated with the higher dose of KCN, no difference was found in T3 or T4 levels between control and dosed pigs, and cholesterol levels were not increased in treated groups. Thus, it is feasible to suppose that alteration in the histology of the thyroid gland and its increase in weight could reect a compensatory

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Figure 2. Liver tissue from a pig that had received 6.0 mg/kg KCN, showing the presence of cariolysis and piknosis in hepatocytes (A) and distortion of the normal lobular architecture of the liver with presence of the deposition of the matriz extra cellular (B). (HE 25)

increase in the efcacy of thyroid hormone synthesis. Therefore, the lack of a toxic effect on the thyroid gland could also be responsible for the unchanged weight gain observed here in the experimental pigs. Chronic cyanide exposure has been associated with a variety of neuronal disorders including tropical ataxic neuropathy, Konzo and ocular pathologies (Osuntokun, 1981; Tucker and Hedges, 1993; Tyllesk ar et al., 1995; Solberg et al., 1998) in humans. However, pathological studies of these neurological diseases are lacking. On the other hand, some natural cases of chronic cyanide exposure in different animals such as lambs (Bradley et al., 1995),

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Figure 3. Kidney tissue from a pig that received 6.0 mg/kg KCN, showing the presence of degeneration of renal tubular epithelial cells, and necrosis. (HE 25)

Figure 4. Degeneration of Purkinje cells of a growing-nishing pig that received 6.0 mg/kg KCN. (HE 25)

horses (Adams et al., 1969) and cattle (McKenzie and McMicking, 1977) fed sorghum are available in the literature. Experimental reproduction of prolonged cyanide exposure was also performed in mice (Mills et al., 1999), rats (Philbrick et al., 1977; Soto-Blanco and Gorniak, 2004) and goats (Soto-Blanco et al., 2002). However, to our knowledge, there are no studies associating prolonged cyanide exposure with neurotoxic effects in pigs. The present data showed that animals treated with KCN presented lesions such as degeneration

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Figure 5. Attenuation of cerebellar white matter of a growing-nishing pig that received 6.0 mg/kg KCN. (HE 25)

of Purkinje cells and loss of cerebellar white matter similar to those reported for rats (SotoBlanco and Gorniak, 2004). Considering that the pig has been extensively used in biomedical research because of the similarities between humans and swine with regard to anatomy and physiology, especially concerning the digestive tract (Ruelius, 1987; Mandel et al., 2004), we suggest that pigs can be used as a new animal model to study some human neuropathies associated with chronic cyanide exposure. The detection of the nephrotoxic effects of cyanide dates back to 1936, when Clark (1936) linked nephrosis to chronic consumption of cassava. Later studies carried out in rats (Ononogbu and Emole, 1978) and dogs (Kamalu, 1993) also associated chronic cassava ingestion and the nephrotoxic effects observed in these animals. In a study on dogs (Kamalu, 1993), hepatotoxicity was also observed and the authors proposed that linamarin was responsible for both pathologies. However, it was only recently that that chronic cyanide consumption was unequivocally shown to have deleterious effects on liver and kidney in rabbits (Okolie and Osagie, 1999) and rats (Sousa et al., 2002). The present study not only conrms the histopathological ndings described in former studies, showing caryolysis and pyknosis in hepatocytes, distortion of the normal lobular architecture of the liver, and degeneration of the renal tubular epithelial cells in experimental pigs, but also reveals impairment of liver and kidney function detected by biochemical evaluation. Since in this experiment KCN was used directly, we may suggest that the liver injury and nephrosis occurring with chronic cassava administration could be produced by cyanide and/or its metabolites, and not by the cyanogenic glycosides linamarin and lotaustralin, or any other compound from cassava. Tropical pancreatic diabetes, also known as tropical diabetes or J diabetes (J from Jamaica, where it was described for the rst time) has been associated with chronic cyanide exposure through consumption of cassava in humans. Its aetiology is still unknown, but

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cyanide exposure through prolonged cassava consumption has been suggested to be responsible (McMillan and Geevarghese, 1979). In a study carried out in dogs fed for 14 weeks on a diet containing cassava, Kamalu (1991) observed an increase in gluconeogenesis from protein as well as hypoinsulinaemia and lesions in the islets of Langerhans and in the acinar portion of the pancreas. On the other hand, in the present study there were no signicant alterations in glucose concentrations or pancreatic morphology in any group of pigs. The lack of a diabetogenic effect observed in the present study is in agreement with results of previous studies in which rats (Sousa, 2000; Soto-Blanco et al., 2001), rabbits (Okolie and Osagie, 2000) and goats (Soto-Blanco et al., 2001) were subjected to prolonged KCN administration. This absence of pancreatic lesions might be explained by two hypotheses. The rst is that humans, but not pigs, rats, rabbits and goats, do develop pancreatic toxicity, which could be related to some feature of cyanide metabolism in these species. A second, and more likely, possibility is that it is the major cyanogenic glucoside of cassava, linamarin, and not cyanide itself, which induces pancreatic toxicity. More experiments are needed to evaluate the supposed link between cyanogenic glucosides and diabetes. In conclusion, the present data permit us to suggest that the constant presence of low concentrations of cyanide in the ration probably has no severe impact on the pig industry. However, this study also revealed that long-term administration of cyanide to pigs promotes neurotoxic, hepatotoxic and nephrotoxic effects, while the thyroid gland and pancreas remained unaffected. Thus, swine could be proposed as an animal model for assessing the chronic toxicity of cyanide to the CNS, liver and kidney; on the other hand, swine are probably a poor model for assessing the goitrogenic effect of cyanide. Whether or not swine are an appropriate animal species for the assessment of the diabetogenic effects of prolonged exposure to cyanide is not known; rst it should be claried whether, in fact, cyanide has toxic effects at the level of the pancreas.

ACKNOWLEDGEMENTS The authors thank Elaine Lanzone, Leonila E.R. Raspantini, Paulo C. Raspantini, Adilson Baladore, Estev ao Belloni and Marco Faustino for technical assistance, and Dr Claudio Alvarenga for the T3 and T4 determinations. This work is part of the Masters thesis presented by Helena Manzano to the Department of Pathology, School of Veterinary Medicine, University of S ao Paulo, Brazil, supported by FAPESP.

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