intoxication by cyanide in pregnant sows: prenatal and ...€¦ · intoxication by cyanide in...

9
Research Article Intoxication by Cyanide in Pregnant Sows: Prenatal and Postnatal Evaluation André T. Gotardo, 1 Isis M. Hueza, 2 Helena Manzano, 1 Viviane M. Maruo, 3 Paulo C. Maiorka, 1 and Silvana L. Górniak 1 1 Research Center of Veterinary Toxicology (CEPTOX), Department of Pathology, School of Veterinary Medicine and Animal Science, University of S˜ ao Paulo, 13635-900 Pirassununga, SP, Brazil 2 Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University of S˜ ao Paulo (ICAQF-UNIFESP), Campus Diadema, 09913-030 Diadema, SP, Brazil 3 College of Veterinary Medicine and Animal Science, Federal University of Tocantins, BR 153, Rural Zone Km 112, 77804-970 Araguaina, TO, Brazil Correspondence should be addressed to Silvana L. G´ orniak; [email protected] Received 7 February 2015; Revised 15 May 2015; Accepted 18 May 2015 Academic Editor: Steven J. Bursian Copyright © 2015 Andr´ e T. Gotardo et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Cyanide is a ubiquitous chemical in the environment and has been associated with many intoxication episodes; however, little is known about its potentially toxic effects on development. e aim of this study was to evaluate the effects of maternal exposure to potassium cyanide (KCN) during pregnancy on both sows and their offspring. Twenty-four pregnant sows were allocated into four groups that orally received different doses of KCN (0.0, 2.0, 4.0, and 6.0 mg/kg of body weight) from day 21 of pregnancy to term. e KCN-treated sows showed histological lesions in the CNS, thyroid follicle enlargement, thyroid epithelial thickening, colloid reabsorption changes, and vacuolar degeneration of the renal tubular epithelium. Sows treated with 4.0mg/kg KCN showed an increase in the number of dead piglets at birth. Weaned piglets from all KCN-treated groups showed histological lesions in the thyroid glands with features similar to those found in their mothers. e exposure of pregnant sows to cyanide thus caused toxic effects in both mothers and piglets. We suggest that swine can serve as a useful animal model to assess the neurological, goitrogenic, and reproductive effects of cyanide toxicosis. 1. Introduction Cyanide is a ubiquitous chemical in the environment and has been associated with many intoxication episodes in human and animals. Cyanide can be released by industrial processes including metal processing, electroplating, and plastic and chemical synthesis [13]. Considering only United States, it is produced 300,000 t of this substance, a year, to feed their industries of electroplating, paper, and plastic and in the extraction of gold [4]. is ion is also present in tobacco smoke [5], and smoke inhalation is a common cause of cya- nide poisoning during fires [6]. Some drugs of medicinal importance, such as laetrile and nitroprusside can release cyanide [7]. Moreover, many plants for human and animal feed, such as Manihot sp. (cassava), Linum sp., Lotus sp., Phaseolus lunatus, and Sorghum sp., contain cyanogenic gly- cosides that release cyanide. e concentration of this sub- stance can be as high as 100–800 mg/kg of plant material [3, 8]. e mechanism of acute cyanide intoxication is well known; it occurs through the inactivation of mitochondrial cytochrome c and disruption of aerobic respiration, which results in potentially fatal cellular hypoxia and cytotoxic anoxia [9]. However, given the constant presence of cyanide in the environment, the most important adverse effects of this compound are currently assumed to be due to chronic toxicity. us, there is a need to carefully assess the potential toxic effects from prolonged exposure to cyanide. Indeed, neuropathies have been attributed to prolonged exposure to low concentrations of cyanide. In humans, Hindawi Publishing Corporation Journal of Toxicology Volume 2015, Article ID 407654, 9 pages http://dx.doi.org/10.1155/2015/407654

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Research ArticleIntoxication by Cyanide in Pregnant Sows Prenatal andPostnatal Evaluation

Andreacute T Gotardo1 Isis M Hueza2 Helena Manzano1 Viviane M Maruo3

Paulo C Maiorka1 and Silvana L Goacuterniak1

1Research Center of Veterinary Toxicology (CEPTOX) Department of Pathology School of Veterinary Medicine andAnimal Science University of Sao Paulo 13635-900 Pirassununga SP Brazil2Institute of Environmental Chemical and Pharmaceutical Sciences Federal University of Sao Paulo (ICAQF-UNIFESP)Campus Diadema 09913-030 Diadema SP Brazil3College of Veterinary Medicine and Animal Science Federal University of Tocantins BR 153 Rural Zone Km 11277804-970 Araguaina TO Brazil

Correspondence should be addressed to Silvana L Gorniak gorniakuspbr

Received 7 February 2015 Revised 15 May 2015 Accepted 18 May 2015

Academic Editor Steven J Bursian

Copyright copy 2015 Andre T Gotardo et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Cyanide is a ubiquitous chemical in the environment and has been associated with many intoxication episodes however little isknown about its potentially toxic effects on development The aim of this study was to evaluate the effects of maternal exposure topotassium cyanide (KCN) during pregnancy on both sows and their offspring Twenty-four pregnant sows were allocated into fourgroups that orally received different doses of KCN (00 20 40 and 60mgkg of body weight) from day 21 of pregnancy to termThe KCN-treated sows showed histological lesions in the CNS thyroid follicle enlargement thyroid epithelial thickening colloidreabsorption changes and vacuolar degeneration of the renal tubular epithelium Sows treated with 40mgkg KCN showed anincrease in the number of dead piglets at birth Weaned piglets from all KCN-treated groups showed histological lesions in thethyroid glands with features similar to those found in their mothers The exposure of pregnant sows to cyanide thus caused toxiceffects in bothmothers and pigletsWe suggest that swine can serve as a useful animal model to assess the neurological goitrogenicand reproductive effects of cyanide toxicosis

1 Introduction

Cyanide is a ubiquitous chemical in the environment and hasbeen associated with many intoxication episodes in humanand animals Cyanide can be released by industrial processesincluding metal processing electroplating and plastic andchemical synthesis [1ndash3] Considering only United States itis produced 300000 t of this substance a year to feed theirindustries of electroplating paper and plastic and in theextraction of gold [4] This ion is also present in tobaccosmoke [5] and smoke inhalation is a common cause of cya-nide poisoning during fires [6] Some drugs of medicinalimportance such as laetrile and nitroprusside can releasecyanide [7] Moreover many plants for human and animalfeed such as Manihot sp (cassava) Linum sp Lotus sp

Phaseolus lunatus and Sorghum sp contain cyanogenic gly-cosides that release cyanide The concentration of this sub-stance can be as high as 100ndash800mgkg of plant material[3 8]

The mechanism of acute cyanide intoxication is wellknown it occurs through the inactivation of mitochondrialcytochrome c and disruption of aerobic respiration whichresults in potentially fatal cellular hypoxia and cytotoxicanoxia [9] However given the constant presence of cyanidein the environment the most important adverse effects ofthis compound are currently assumed to be due to chronictoxicity Thus there is a need to carefully assess the potentialtoxic effects from prolonged exposure to cyanide

Indeed neuropathies have been attributed to prolongedexposure to low concentrations of cyanide In humans

Hindawi Publishing CorporationJournal of ToxicologyVolume 2015 Article ID 407654 9 pageshttpdxdoiorg1011552015407654

2 Journal of Toxicology

chronic and spontaneous degenerative diseases such as spas-tic paraparesis or ldquokonzordquo [10] and tropical ataxic neuropathy[11 12] have been associated with high cassava consumptionFurthermore prolonged cyanide exposure has been associ-ated with Parkinsonrsquos disease and cognitive impairment [1314]

Tobacco smoking and cassava consumption have beenimplicated in the pathogenesis of ocular diseases such astobacco-alcohol amblyopia [15 16] retrobulbar neuropathyof pernicious anemia [17] Leberrsquos hereditary optic neuropa-thy [18 19] West Indian amblyopia [20 21] Jamaican opticneuropathy [22] tropical amblyopia [23] and Cuban opticneuropathy [24]

In addition pancreatic and tropical diabetes in humansand animals have been associated with the ingestion ofcyanogenic plants [25 26] especially cassava [27]

Experimental studies in dogs goats and rats have shownthat chronic cyanide exposure causes impaired body growthgoiter and lesions of the central nervous system (CNS) [28ndash30] In addition we have verified that the long-term admin-istration of cyanide to growing pigs promotes neurotoxichepatotoxic and nephrotoxic effects [31]

Although studies have been conducted on chronic cya-nide intoxication in various animal species little is knownabout its potentially toxic effects on development Fetalmalformations have been linked to maternal consumptionof cyanogenic plants in animals such as pigs horses sheepand cattle [32ndash36] as well as in humans [37 38] Moreoverstudies of goats conducted in our lab have shown thatmaternal cyanide exposure is associated with fetal abortionand retrognathia [39] We verified these findings in studieswith rats where pups from dams treated with different dosesof cyanide andor thiocyanate (the activemetabolite resultingfrom the biotransformation of cyanide) exhibited similartoxic effects some of which were observed only at weaning[40]

We have previously demonstrated that the pig is a usefulanimal model for assessing chronic cyanide toxicity [31]and others have found it suitable for reproductive toxicologystudies [41 42] The aim of this study was to evaluate thetoxic effects in both dams and their offspring of maternalexposure to potassium cyanide (KCN) during pregnancy inpigs

2 Materials and Methods

This study was conducted at the University of Sao Paulo(USP) Experimental Station Pirassununga SP Brazil(S21∘581015840 W47∘271015840) The procedures were approved by theUSP Animal Ethics Committee and all animal care and han-dling was performed by experienced personnel under veter-inary supervision

21 Animals Feeding and Experimental Design Landrace-Large White sows 240 days old were bred to one male pigof the same breed All females were negative for antibodies toAujeszkyrsquos disease and for infections with Campylobacter spMycobacterium avium Brucella suis and Leptospira sp

The day of breeding was defined as gestational day 1(GD1) and pregnancies were confirmed by ultrasound (US)apparatus (Scanner 100 Vet Pie Medical) on GD21 Afterthis confirmation females began to receive the experimentalrations Pregnant sows were randomly allocated into fourtreatment group (119873 = 6 per group at the beginning of theexperiment) and administered varying doses of KCN (MerckGermany) in mg per kg of body weight (mgkg BW) asfollows 0 (KCN0 group) 20 (KCN2 group) 40 (KCN4group) and 60 (KCN6 group)

The purpose of this study was to assess for embryotoxiceffects of cyanide using swine as amodel organismGiven thatembryo implantation in swine starts on day 13 after breedingand ends on day 18 [43] KCN was administered from GD21to parturition (approximately day 114 of pregnancy) twice aday with one half dose administered between 700 and 0730and the other half dose administered between 1730 and 1800mixed with 10 g of starch biscuits in individual troughs Thecontrol group received only the biscuitsWater and food werefreely available During gestation the dams were weighedweekly

22 Biochemical and Hormonal Evaluations Blood sampleswere collected via jugular venipuncture using heparinizedsyringes at the 21st 50th 80th and 110th days of pregnancyBlood serum was frozen and stored at minus10∘C until analysisCommercial kits (CELM Brazil) were used for the determi-nation of albumin glucose urea creatinine aspartate amino-transferase (AST) and gamma glutamyltranspeptidase(GGT) levels using automatic biochemical system (SBA-200CELM) The plasmatic concentrations of thyroxine (T

4)

and triiodothyronine (T3) were measured on GD110 using

commercial radioimmunoassay kits (Coat-A-Count Siem-ens)

23 Ultrasonographic Evaluation and Pathological Study inSows Ultrasonographic evaluationwas performed onGD45GD55 and GD65 in each pregnant swine to measure the fol-lowing fetal parameters crown-rump length (CRL) thoracicdiameter (TD) abdominal diameter (AD) and biparietaldiameter (BPD)

On GD110 one pregnant sow from each group was euth-anized the amniotic fluid was collected for chemical analysisFragments of the CNS thyroid glands lungs pancreasliver and kidneys were collected and fixed in 10 bufferedformalin embedded in paraffin cut into 5120583m sections andstained by hematoxylin and eosin (HE)

24 Evaluations in Piglets Immediately at birth each neo-natersquos body weight and gender were recorded Each newbornwas then examined carefully for gross abnormalities [44]Two newborn pigs a male and female from each motherwere then euthanized to collect fragments of the CNSthyroid glands lungs pancreas liver and kidneys Frombirth(PND1) to three months of age (PND120) each piglet wasweighed weekly and the weight gains were calculated Theabsolute weights andweight gains of the piglets were analyzed

Journal of Toxicology 3

separately by gender Blood samples from each piglet weretaken via jugular venipuncture immediately after birth and onthe 7th 14th 21st and 45th days of life for the evaluation ofthe same biochemical parameters assessed in their mothers

At the end of the experimental period (PND120) thepiglets were euthanized Fragments of the CNS thyroidpancreas liver and kidney were collected and fixed in 10buffered formalin embedded in paraffin cut into 5 120583msections and stained by hematoxylin and eosin (HE)

25 Analytical Procedures Thiocyanate levels of gilts weredosed in serum at GD21 GD50 GD80 and GD110 in amni-otic fluid on GD110 and into colostrum immediately afterparturition by spectrophotometry using the method outlinedby Pettigrew and Fell [15] with modifications specific to ourstudy Briefly 02mL of the biologicalmaterials were added to18mL of a 10 trichloroacetic acid solution and centrifugedat 10000 rpm for 20minNext 05mLof supernatant aliquotswas acidified with 025mL of 1M HCl The samples weremixed after adding each of the following reagents 50 120583L ofsaturated bromine water 100 120583L of trioxide arsenious (20 gLin 01 NNaOH) 09mLof pyridine (10mLof 12NHCl 60mLof pyridine and 40mL of deionized water) and p-PDAreagent (2 g in 1 L of 05M HCl 3 1 prepared immediatelybefore use) The reddish-pink complex that formed was readat 540 nm after 15min in a spectrophotometer (MicronalB382 Micronal SA Brazil) against a blank sample Thio-cyanate levels in the samples were read based on standardcurves prepared with thiocyanate of known concentrations(25ndash300 120583molL) Data were expressed as 120583LmolL

The linearity of the analytical procedure was tested atconcentrations of 25 50 75 100 200 and 300120583molLof thiocyanate in 10 samples for each concentration Thecoefficient of determination was 09911 slope 00008097and the 119910-axis intercept in 0005267 The intralaboratoryprecision expressed by the coefficient of variation intra- andinterday was obtained by analyzing the levels of thiocyanatein standard samples of 100120583molL in water and pool samplesof serum colostrum and amniotic fluid processed fivetimes each on two different days The coefficient of variationranged from 13 to 58 intra- and 21 to 48 interday Thethiocyanate recovery was from 955 to 983 for serum 945 to966 for colostrum and 988 to 982 for amniotic fluid addedto the standard solution of thiocyanate at concentrations of100 and 200120583molL respectively

26 Statistical Analysis Weight gain data serum biochem-istry ultrasound measurements and serum thiocyanate lev-els were analyzed using a mixed linear model (Proc Mixed)for each treatment The animals were nested within thetreatments and repeated measurements of the variables weretaken over time The animals were considered a randomfactor in the model

Hormone serum levels birth weights and thiocyanatelevels in the colostrumwere analyzed statistically by one-wayanalysis of variance (ANOVA) followed byDunnettrsquos testThefrequency of dead piglets was analyzed using Fisherrsquos exacttest with each treatment compared to the controls

Data are reported as the means plusmn SEM and were analyzedusing SAS software (Version 92 SAS Institute Cary NC) Inall cases the probability of significant differences was set at120572 = 005

Calibration and concentration curves of thiocyanate inthe different biological fluids versus the time taken aftertreatments were made using the GraphPad Prism 500(Graphpad 2007)

3 Results

Pregnant sows did not show clinical signs of acute cyanideintoxication during the experimental period All the damssurvived the treatment throughout the study There wasno significant difference between controls and KCN-treatedsows in body weight gain (Table 1 119875 gt 005)

Fetal deaths were diagnosed by ultrasonographic eval-uation in three females from the KCN4 group and in onepregnant female from KCN6 group between 21 and 45 daysof gestation Females that failed pregnancy were withdrawnfrom the experiment and replaced with pregnant females

Biochemical analysis of serum collected during preg-nancy showed fluctuations of multiple parameters but theseparameters remained within the normal ranges for all swine(data not shown) Similarly no changes in T

3(control 0039 plusmn

0005 KCN2 0033 plusmn 0001 KCN4 0034 plusmn 0002 andKCN6 0034 plusmn 0002) and T

4(control 084 plusmn 04 KCN2

09 plusmn 03 KCN4 079 plusmn 01 and KCN6 093 plusmn 02) levelswere found on the 110th day of gestation On the otherhand plasma thiocyanate levels were increased on GD50GD80 and GD110 in females from all treated groups (Fig-ure 1 119875 lt 005) Colostrum concentrations of thiocyanatewere increased in all dams treated with cyanide (Figure 1119875 lt 005) Amniotic fluid evaluated from one female ineach group showed increased thiocyanate levels in a dose-response fashion (KCN0 205120583molL KCN2 511 120583molLKCN4 559 120583molL and KCN6 657 120583molL)

Ultrasonographic evaluation at GD45 GD55 and GD65demonstrated no significant changes in the morphometricmeasurements (CRL TDAD andBPD) of the fetuses in eachgroup (data not shown)

In histopathological assessments of euthanized pregnantsows at GD110 a greater number of Purkinje cells withacidophilic cytoplasm were found in all experimental sowswhereas vacuolar degeneration of Purkinje cells was observedonly in sows from the KCN6 group (Figure 2) Compared tothe control sows the experimental sows had thyroid follicleenlargement thickening of the cubic follicular epitheliumand reabsorption vacuoles in colloid (Figure 3) Additionallythere was vacuolar degeneration of the renal tubular epithe-lium in sows from all groups exposed to KCN (Figure 4)

There was a significant increase (119875 = 0002) in the num-ber of dead piglets in the KCN4 group compared with theKCN0 group (Table 1) The body weight of the piglets at birthwas unaffected (Table 1 119875 gt 005) by the treatment

The biochemical analysis of serum collected from pigletsshowed fluctuations of the different parameters but theseparameters remained within the normal ranges for swine

4 Journal of Toxicology

Table 1 Reproductive parameters from sows treated with KCN from day 21 of pregnancy to term and the body weight of their piglets at birthuntil 120 days old

Parameters Control (119899 = 6) KCN (mgkg per day)20 (119899 = 6) 40 (119899 = 6) 60 (119899 = 6)

Body weight gain (kg) during pregnancya 696 plusmn 46 552 plusmn 51 498 plusmn 41 594 plusmn 82Number of assisted births 0 (5)b 0 (5) 4 (5) 1 (5)Live piglets 57 55 36 51

Male 27 30 13 24Female 30 25 23 27

Number of dead piglets 6 4 23lowast 10

Stillbirths 5 4 14 7Mummified fetuses 1 0 9 3

Subsequent total born piglets (mean) 126 118 72 94Piglets body weight at birtha 168 plusmn 004 (57)c 164 plusmn 003 (55) 171 plusmn 007 (36) 160 plusmn 005 (51)Overall weight gain (kg) of piglets at 120 days

Males 5525 plusmn 382 5247 plusmn 243 5407 plusmn 297 5279 plusmn 349Females 5382 plusmn 324 5235 plusmn 278 5162 plusmn 197 5162 plusmn 299

aMean plusmn SEMbNumber of deliveriescNumber of piglets weighedlowast

119875 lt 005 compared with controls

0

50

100

150

200

250

KCN0KCN2

KCN4KCN6

21 50 80 110Days of pregnancy

Thio

cyan

ate (

120583m

olL

)

(a)

KCN0KCN2

KCN4KCN6

0

10

20

30

40

50

Colostrum

Thio

cyan

ate (

120583m

olL

)

(b)

Figure 1 Thiocyanate levels (means plusmn SEM) in plasma (a) and colostrum (b) of sows allocated into four treatment groups KCN0 KCN2KCN4 andKCN6 that received respectively 00 20 40 and 60mgkg of bodyweight of cyanide fromday 21 of pregnancy to term 119901 lt 005compared to control

(data not shown) At PND120 there was no treatment effecton the overall weight gain of male or female piglets (Table 1)No histopathological lesions were observed in euthanizednewborns At PND120 piglets in all experimental groups hadsimilar histopathological changes as their mothers includingthyroid follicular enlargement and epithelial thickening andvacuolization (Figure 3)

4 Discussion

The administration of KCN orally was chosen aiming tomimic the main route of natural exposure to this speciesThedoses of KCN used here were chosen based on data from anearlier study conducted in our laboratory on male growing-finishing swine [31] inwhich the animals showed toxic effects

Journal of Toxicology 5

(b) (c)

(a)

Figure 2 Light photomicrograph of pregnant sows CNS (a) animal from control group ((b) and (c) resp) animals treated with 40 and60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing Purkinje cells with acidophilic cytoplasm (arrow (b)) andvacuolar degeneration of Purkinje cells (arrow (c)) Magnification times20 HE

(a) (b)

(c) (d)

Figure 3 Light photomicrograph of pregnant sows thyroid ((a) and (c)) animals from control group ((b) and (d)) animals treated with60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing enlargement of thyroid follicles (b) thickening of thefollicular epithelium (arrow (d)) and reabsorption vacuoles in colloid (arrowhead (d)) Magnification times4 ((a) and (b)) times20 ((c) and (d))HE

of chronic exposure to cyanide but did not manifest anyclinical signs of acute toxicity (eg dyspnea and convulsiveseizures) Similarly none of the pregnant females in thisstudy showed clinical symptoms of acute cyanide intoxicationduring the period of KCN administration

Given that the bitter taste of cyanide could compromisefood consumption during the study [45] starch biscuitsknown to be highly palatable to swine were chosen toborne the cyanide This procedure ensured that all femalesingested the target doses of cyanide quickly (usually in lessthan 2min) in each administration In a previous study oftoxicokinetics in pregnant sows we verified that following

oral exposure to KCN serum thiocyanate levels increase by3 hrs peak after 6 hrs and remain elevated for up to 21 hrs[46] Thus we assumed that the administration of cyanidetwice daily would ensure high serum levels of thiocyanatesteadily independently of the phase of pregnancy

Although some studies have found an associationbetween the reduction of body weight and prolonged expo-sure to cyanide in humans and animals [47ndash49] we did notdetect a decrease in this parameter in any of the pregnant sowsexposed to KCN This result corresponds with our previouswork conducted in growing-finishing pigs [31] Likewisestudies evaluating cyanide exposure during pregnancy in

6 Journal of Toxicology

(a)

(b) (c)

Figure 4 Light photomicrograph of pregnant sows kidney (a) animal from control group ((b) and (c) resp) animals treated with 20 and60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing vacuolar degeneration of the renal tubular epithelium(arrow (b) and (c)) Magnification times20 HE

goats [39] and rats [40] have not found differences in weightgain between the control and experimental animal groups Inthe same manner Tewe and Maner [50] did not find weightchanges in pregnant pigs fed cassava containing high levels ofcyanide

Although no biochemical changes were observed in preg-nant females exposed to KCN the histopathology performedin these sows revealed kidney and liver damage Lesionssimilar to those observed in our study have previously beendescribed in studies of other species chronically exposed tocyanide [30 51 52] as well as in pigs [31] It remains tobe determined if these lesions are a consequence of toxicitydirectly from cyanide thiocyanate or both

Studies clearly show that the CNS is an important targetin prolonged exposure to cyanide in both humans [53] andanimals [54ndash56] In humans it has been described to resultin several neuropathies such as spastic paraparesis (alsocalled ldquokonzordquo) Parkinsonrsquos disease cognitive impairmentand ocular pathologies [11 13 14 16 24 57]

The pig is an emerging model system for studying thehuman CNS [58] In a previous study in our lab where cya-nide was administered to growing pigs during the sameperiod evaluated here we found Purkinje cell degenerationand loss of cerebellar white matter in all groups treatedwith KCN [31] Similar lesions were found in this studyin sows exposed to cyanide These findings strengthen ourassertion that pigs should be used as an animal model tobetter understand the mechanism(s) of neurotoxicity fromcyanide andor its main metabolite

Tropical pancreatic diabetes has been associated withchronic cyanide exposure through consumption of cassavain man [25] and was reproduced in studies of dogs fedcassava [59] However studies administering cyanide todifferent animal species [29 60] including pigs [31] were

not able to reproduce these diabetogenic effects despite theanimals having islet cells functionality similar to humans[61] Given that pregnancy itself has a diabetogenic effectdue to a decrease in insulin sensitivity of maternal tissuesand resultant increase in insulin demand [62 63] pregnantsows would theoretically be evenmore susceptible to developdiabetes upon cyanide exposure Thus we hypothesized thatour study would have offered all the conditions needed toreproduce tropical pancreatic diabetes in pigs However wedid not find any changes in the parameters chosen to evaluatethe diabetogenic effects of cyanide (ie glucose levels andhistopathology) As we have previously shown that pregnantrats exposed to cyanide have increased levels of glucose andpancreatic islet degeneration [40] pregnant rats may be abetter animal model to assess the pancreotoxic effects ofcyanide

Studies have found a correlation between low doses ofchronic cyanide intake with a higher incidence of abortion inmares [32] sheep [36] and goats [39] Chronic cyanide intakein low doses from tobacco smoke has similarly been associ-ated with spontaneous abortion in women [64] although itremains unclear if this finding is specifically due to cyaniderather than other toxic compounds found in cigarettes

Higher rates of fetal death stillbirth assisted birth andmummified piglets were found in sows exposed to cyanidein this study The most common infectious causes of repro-ductive disease in Brazil (Campylobacter sp Mycobacteriumavium Brucella suis and Leptospira sp) were ruled out inthese animals Many causes could be posited to explain thesefinding but it is plausible to consider KCN as the mostlikely one We therefore suggest that the pig is a good animalmodel system for studying the role of cyanide andor its mainmetabolite in smoking-related abortions in women In ourstudy as we did not detect malformations in newborn pigs

Journal of Toxicology 7

it was not possible to conclude if ultrasound evaluation inpigs could be valuable for early detection of malformationsldquoin uterordquo

Amniocenteses in this study revealed high levels ofthiocyanate in the amniotic fluid of pregnant sows Denckeret al [65] and Schwartz [66] show that anionic substancessuch as the weak acid cyanuric acid can accumulate in theembryo and fetus This may be due to the pH gradient inmaternal plasma which is more acidic than the pH of fetalplasma Studies have thus observed an ldquoimprisonmentrdquo ofcyanide in the fetal organism [67] In addition it is knownthat the enzyme rhodanese is found in the placenta and thefetus [68] Thus it is possible that the ldquoarrestedrdquo cyanidecan be transformed into its main metabolite resulting inindirect thiocyanate toxicity to the fetus This hypothesisis additionally supported by a study in which cyanide orthiocyanate was administered to pregnant rats and bothsubstances were found to promote toxic effects in the fetuses[40]

The protocols that have been used to assess develop-mental toxicity in previous studies have certain limitations[69] One such limitation is the lack of postnatal monitoringof the development of the offspring which can result inerroneous conclusions about the developmental toxicity ofthe substances under examination In fact de Sousa et al [40]evaluated prenatal exposure to cyanide in rats and found thatalterations in pups were detected only at weaning Our studysupports these findings as we identified certain toxic effectsof cyanide in the piglets only at weaning

5 Conclusion

In conclusion pregnant sows exposed to cyanide had toxiceffects in both mothers and piglets However the toxiceffects in the offspring were observed belatedly Thereforethe data presented here reinforces the hypothesis that acomplementary evaluation of the neonate after weaning is anecessary component of studies on developmental toxicityIn addition we suggest that swine can serve as a usefulanimal model to study the neurological goitrogenic andreproductive toxicity of cyanide

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] W C Beamer R M Shealy and D S Prough ldquoAcute cyanidepoisoning from laetrile ingestionrdquo Annals of Emergency Medi-cine vol 12 no 7 pp 449ndash451 1983

[2] R Froldi M Cingolani and C Cacaci ldquoA case of suicide byingestion of sodium nitroprussiderdquo Journal of Forensic Sciencesvol 46 no 6 pp 1504ndash1506 2001

[3] J E Poulton ldquoCyanogenic compounds in plants and their toxiceffectsrdquo inHandbook of Natural Toxins Plant and Fungal ToxinsR F Keeler and A R Tu Eds vol 1 pp 117ndash157 Marcel DekkerNew York NY USA 1983

[4] United States Army Medical Research Institute of ChemicalDefense and Chemical Casualty Care Division ldquoCyaniderdquo inMedicalManagement of Chemical Causalities Handbook UnitedStates Army Medical Research Institute of Chemical DefenseAberdeen Md USA 3rd edition 2004

[5] M J Koschel ldquoWhere therersquos smoke there may be cyaniderdquoTheAmerican Journal of Nursing vol 102 no 8 pp 39ndash42 2002

[6] K Anseeuw N Delvau G Burillo-Putze et al ldquoCyanide poi-soning by fire smoke inhalation a European expert consensusrdquoEuropean Journal of Emergency Medicine vol 20 no 1 pp 2ndash92013

[7] V Schulz ldquoClinical pharmacokinetics- of nitroprusside cya-nide thiosulphate and thiocyanaterdquo Clinical Pharmacokineticsvol 9 no 3 pp 239ndash251 1984

[8] J Vetter ldquoPlant cyanogenic glycosidesrdquo Toxicon vol 38 no 1pp 11ndash36 2000

[9] B Ballantyne ldquoToxicology of cyanidesrdquo in Clinical and Exper-imental Toxicology of Cyanides B Ballantyne and T C MarrsEds pp 41ndash126 Wright Bristol UK 1987

[10] H Nzwalo and J Cliff ldquoKonzo from poverty cassava andcyanogen intake to toxico-nutritional neurological diseaserdquoPLoS Neglected Tropical Diseases vol 5 no 6 Article ID e10512011

[11] B O Osuntokun ldquoCassava diet chronic cyanide intoxicationand neuropathy in the Nigerian Africansrdquo World Review ofNutrition and Dietetics vol 36 pp 141ndash173 1981

[12] J Njoh ldquoTropical ataxic neuropathy in Liberiansrdquo Tropical andGeographical Medicine vol 42 no 1 pp 92ndash94 1990

[13] N L Rosenberg J A Myers and W R Wayne Martin ldquoCya-nide-induced parkinsonism clinical MRI and 6-fluorodopaPET studiesrdquo Neurology vol 39 no 1 pp 142ndash144 1989

[14] M Di Filippo N Tambasco G Muzi et al ldquoParkinsonism andcognitive impairment following chronic exposure to potassiumcyaniderdquoMovement Disorders vol 23 no 3 pp 468ndash470 2008

[15] A R Pettigrew and G S Fell ldquoSimplified colorimetric determi-nation of thiocyanate in biological fluids and its application toinvestigation of the toxic amblyopiasrdquo Clinical Chemistry vol18 no 9 pp 996ndash1000 1972

[16] Y Solberg M Rosner and M Belkin ldquoThe association betwencigarette smoking and ocular diseasesrdquo Survey of Ophthalmol-ogy vol 42 no 6 pp 535ndash547 1998

[17] A G Freeman ldquoOptic neuropathy and chronic cyanide intox-ication a reviewrdquo Journal of the Royal Society of Medicine vol81 no 2 pp 103ndash106 1988

[18] I G Syme J Bronte-Stewart and W S Foulds ldquoClinical andbiochemical findings in Leberrsquos hereditary optic atrophyrdquoTrans-actions of the Ophthalmological Societies of the United Kingdomvol 103 no 5 pp 556ndash559 1983

[19] K Tsao P A Aitken and D R Johns ldquoSmoking as an aetio-logical factor in a pedigree with Leberrsquos hereditary optic neu-ropathyrdquo British Journal of Ophthalmology vol 83 no 5 pp577ndash581 1999

[20] S J Crews ldquoBilateral amblyopia in west Indiansrdquo Transactionsof the Ophthalmological Societies of the United Kingdom vol 83pp 653ndash667 1963

[21] A D Mackenzie and C I Phillips ldquoWest Indian amblyopiardquoBrain vol 91 no 2 pp 249ndash260 1968

[22] F D Carroll ldquoJamaican optic neuropathy in immigrants to theUnited StatesrdquoThe American Journal of Ophthalmology vol 71no 1 pp 261ndash265 1971

8 Journal of Toxicology

[23] B O Osuntokun and O Osuntokun ldquoTropical amblyopia inNigeriansrdquo American Journal of Ophthalmology vol 72 no 4pp 708ndash716 1971

[24] K Tucker and T R Hedges ldquoFood shortages and an epidemicof optic and peripheral neuropathy in CubardquoNutrition Reviewsvol 51 no 12 pp 349ndash357 1993

[25] D E McMillan and P J Geevarghese ldquoDietary cyanide andtropical malnutrition diabetesrdquo Diabetes Care vol 2 no 2 pp202ndash208 1979

[26] C S Pitchumoni N K Jain A B Lowenfels and E P DimagnoldquoChronic cyanide poisoning unifying concept for alcoholic andtropical pancreatitisrdquo Pancreas vol 3 no 2 pp 220ndash222 1988

[27] B P Kamalu ldquoPathological changes in growing dogs fed ona balanced cassava (Manihot esculenta Crantz) dietrdquo BritishJournal of Nutrition vol 69 no 3 pp 921ndash934 1993

[28] B P Kamalu ldquoThe adverse effects of long-term cassava (Mani-hot esculenta Crantz) consumptionrdquo International Journal ofFood Sciences and Nutrition vol 46 no 1 pp 65ndash93 1995

[29] B Soto-Blanco S L Gorniak and E T Kimura ldquoPhysiopatho-logical effects of the administration of chronic cyanide togrowing goatsmdasha model for ingestion of cyanogenic plantsrdquoVeterinary ResearchCommunications vol 25 no 5 pp 379ndash3892001

[30] A B Sousa B Soto-Blanco J L Guerra E T Kimura and SL Gorniak ldquoDoes prolonged oral exposure to cyanide promotehepatotoxicity and nephrotoxicityrdquo Toxicology vol 174 no 2pp 87ndash95 2002

[31] H Manzano A B de Sousa B Soto-Blanco J L Guerra P CMaiorka and S L Gorniak ldquoEffects of long-term cyanide inges-tion by pigsrdquo Veterinary Research Communications vol 31 no1 pp 93ndash104 2007

[32] J T Prichard and J L Voss ldquoFetal ankylosis in horses associatedwith hybrid Sudan pasturerdquo Journal of the American VeterinaryMedical Association vol 150 no 8 pp 871ndash873 1967

[33] L A Selby R W Menges E C Houser R E Flatt and A ACase ldquoOutbreak of swine malformations associated with thewild black cherry Prunus serotinardquo Archives of EnvironmentalHealth vol 22 no 4 pp 496ndash501 1971

[34] M G W Rodel ldquoEffects of different grasses on the incidence ofneonatal goiter and skeletal deformities in autumn born lambsrdquoRhodesia Agricultural Journal vol 69 pp 59ndash60 1972

[35] J T Seaman M G Smeal and J C Wright ldquoThe possibleassociation of a sorghum (Sorghum sudanese) hybrid as acause of developmental defects in calvesrdquo Australian VeterinaryJournal vol 57 no 7 pp 351ndash352 1981

[36] G A Bradley H C Metcalf C Reggiardo et al ldquoNeuroaxonaldegeneration in sheep grazing Sorghum pasturesrdquo Journal ofVeterinary Diagnostic Investigation vol 7 no 2 pp 229ndash2361995

[37] J D Singh ldquoThe teratogenic effects of dietary cassava on thepregnant albino rat a preliminary reportrdquo Teratology vol 24no 3 pp 289ndash291 1981

[38] H K Kumbnani A Singh G Singh and P Parimoo ldquoLimbrsquosmalformations in SouthernNigeriamdashGarri (Manihot esculenta)a probable causerdquo Journal of Human Ecology vol 1 pp 189ndash1911990

[39] B Soto-Blanco and S L Gorniak ldquoPrenatal toxicity of cyanidein goatsmdasha model for teratological studies in ruminantsrdquoTheriogenology vol 62 no 6 pp 1012ndash1026 2004

[40] A B de Sousa P CMaiorka I DGoncalves L RM de Sa andS L Gorniak ldquoEvaluation of effects of prenatal exposure to the

cyanide and thiocyanate inwistar ratsrdquoReproductive Toxicologyvol 23 no 4 pp 568ndash577 2007

[41] N C Ganderup W Harvey J T Mortensen and W HarroukldquoTheminipig as nonrodent species in toxicologymdashwhere are wenowrdquo International Journal of Toxicology vol 31 no 6 pp 507ndash528 2012

[42] G Bode P Clausing F Gervais et al ldquoThe utility of theminipigas an animal model in regulatory toxicologyrdquo Journal of Phar-macological and Toxicological Methods vol 62 no 3 pp 196ndash220 2010

[43] R D Geisert R H Renegar W W Thatcher R M Robertsand FW Bazer ldquoEstablishment of pregnancy in the pig I Inter-relationships between preimplantation development of the pigblastocyst and uterine endometrial secretionsrdquoBiology of Repro-duction vol 27 no 4 pp 925ndash939 1982

[44] K T Szabo Congenital Malformations in Laboratory and FarmAnimals Academic Press San Diego Calif USA 1989

[45] E A Iyayi ldquoDietary cyanide effect on performance and serumtestosterone of growing male pigsrdquo Beitrage zur TropischenLandwirtschaft undVeterinarmedizin vol 29 no 3 pp 347ndash3521991

[46] H Manzano A B Souza and S L Gorniak ldquoExposicaocianıdrica em suınos uma abordagem dos parametros toxic-ocineticos utilizando o tiocianato como biomarcadorrdquoBrazilianJournal of Veterinary Research and Animal Science vol 43 pp93ndash101 2006

[47] P Blanc M Hogan K Mallin D Hryhorczuk S Hessl and BBernard ldquoCyanide intoxication among silver-reclaiming work-ersrdquo The Journal of the American Medical Association vol 253no 3 pp 367ndash371 1985

[48] C F I Onwuka ldquoHydrocyanic acid contents of tropical browseand their influence on performance of goatsrdquo Food Chemistryvol 45 no 1 pp 5ndash10 1992

[49] C Ibebunjo B P Kamalu and E C Ihemelandu ldquoComparisonof the effects of cassava (Manihot esculenta Crantz) organiccyanide and inorganic cyanide on muscle and bone develop-ment in a Nigerian breed of dogrdquo British Journal of Nutritionvol 68 no 2 pp 483ndash491 1992

[50] O O Tewe and J H Maner ldquoPerformance and pathophysio-logical changes in pregnant pigs fed cassava diets containingdifferent levels of cyaniderdquo Research in Veterinary Science vol30 no 2 pp 147ndash151 1981

[51] N P Okolie and A U Osagie ldquoLiver and kidney lesions andassociated enzyme changes induced in rabbits by chronic cya-nide exposurerdquo Food and Chemical Toxicology vol 37 no 7 pp745ndash750 1999

[52] B Soto-Blanco and S L Gorniak ldquoToxic effects of prolongedadministration of leaves of cassava (Manihot esculenta Crantz)to goatsrdquo Experimental and Toxicologic Pathology vol 62 no 4pp 361ndash366 2010

[53] J Wilson ldquoCyanide in human disease a review of clinical andlaboratory evidencerdquo Fundamental and Applied Toxicology vol3 no 5 pp 397ndash399 1983

[54] E M Mills P G Gunasekar L Li J L Borowitz and G EIsom ldquoDifferential susceptibility of brain areas cyanide involvesdifferent modes of cell deathrdquo Toxicology and Applied Pharma-cology vol 156 no 1 pp 6ndash16 1999

[55] B Soto-Blanco P CesarMarioka and S Lima Gorniak ldquoEffectsof long-term low-dose cyanide administration to ratsrdquo Ecotoxi-cology and Environmental Safety vol 53 no 1 pp 37ndash41 2002

Journal of Toxicology 9

[56] B Soto-Blanco F T V Pereira A F D CarvalhoM AMiglinoand S L Gorniak ldquoFetal andmaternal lesions of cyanide dosingto pregnant goatsrdquo Small Ruminant Research vol 87 no 1ndash3 pp76ndash80 2009

[57] T Tylleskar M Banea N Bikangi G Nahimana L-A Perssonand H Rosling ldquoDietary determinants of a non-progressivespastic paraparesis (Konzo) a case-referent study in a highincidence area of Zairerdquo International Journal of Epidemiologyvol 24 no 5 pp 949ndash956 1995

[58] M M Swindle A Makin A J Herron F J Clubb Jr and K SFrazier ldquoSwine asmodels in biomedical research and toxicologytestingrdquo Veterinary Pathology vol 49 no 2 pp 344ndash356 2012

[59] B P Kamalu ldquoThe effect of a nutritionally-balanced cassava(Manihot esculenta Crantz) diet on endocrine function usingthe dog as a model 1 Pancreasrdquo British Journal of Nutrition vol65 no 3 pp 365ndash372 1991

[60] N P Okolie and A U Osagie ldquoDifferential effects of chroniccyanide intoxication on heart lung and pancreatic tissuesrdquo Foodand Chemical Toxicology vol 38 no 6 pp 543ndash548 2000

[61] M M Swindle and A C Smith ldquoComparative anatomy andphysiology of the pigrdquo Scandinavian Journal of LaboratoryAnimal Science vol 25 no 1 pp 11ndash21 1998

[62] L Jovanovic andD J Pettitt ldquoGestational diabetesmellitusrdquoTheJournal of the AmericanMedical Association vol 286 no 20 pp2516ndash2518 2001

[63] TA Buchanan andAHXiang ldquoGestational diabetesmellitusrdquoThe Journal of Clinical Investigation vol 115 no 3 pp 485ndash4912005

[64] The Practice Committee of American Society for ReproductiveMedicine ldquoSmoking and infertilityrdquo Fertility and Sterility vol4 pp 1181ndash1186 2004

[65] L Dencker R DrsquoArgy B R G Danielsson H Ghantous and GO Sperber ldquoSaturable accumulation of retinoic acid in neuraland neural crest derived cells in early embryonic developmentrdquoDevelopmental Pharmacology andTherapeutics vol 10 no 3 pp212ndash223 1987

[66] S Schwartz ldquoProviding toxicokinetic support for reproductivetoxicology studies in pharmaceutical developmentrdquo Archives ofToxicology vol 75 no 7 pp 381ndash387 2001

[67] W J Scott and H Nau ldquoAccumulation of weak acids in theyoung mammalian embryordquo in Pharmacokinetics in Teratoge-nesis H Nau and W J Scott Eds pp 72ndash76 CRC Press BocaRaton Fla USA 1987

[68] O O Tewe J H Maner and G Gomez ldquoInfluence of cassavadiets on placental thiocyanate transfer tissue rhodanese activityand performance of rats during gestationrdquo Journal of the Scienceof Food and Agriculture vol 28 no 8 pp 750ndash756 1977

[69] L Claudio C F Bearer andDWallinga ldquoAssessment of theUSenvironmental protection agency methods for identification ofhazards to developing organisms part II the developing toxicitytesting guidelinerdquo American Journal of Industrial Medicine vol35 no 6 pp 554ndash563 1999

2 Journal of Toxicology

chronic and spontaneous degenerative diseases such as spas-tic paraparesis or ldquokonzordquo [10] and tropical ataxic neuropathy[11 12] have been associated with high cassava consumptionFurthermore prolonged cyanide exposure has been associ-ated with Parkinsonrsquos disease and cognitive impairment [1314]

Tobacco smoking and cassava consumption have beenimplicated in the pathogenesis of ocular diseases such astobacco-alcohol amblyopia [15 16] retrobulbar neuropathyof pernicious anemia [17] Leberrsquos hereditary optic neuropa-thy [18 19] West Indian amblyopia [20 21] Jamaican opticneuropathy [22] tropical amblyopia [23] and Cuban opticneuropathy [24]

In addition pancreatic and tropical diabetes in humansand animals have been associated with the ingestion ofcyanogenic plants [25 26] especially cassava [27]

Experimental studies in dogs goats and rats have shownthat chronic cyanide exposure causes impaired body growthgoiter and lesions of the central nervous system (CNS) [28ndash30] In addition we have verified that the long-term admin-istration of cyanide to growing pigs promotes neurotoxichepatotoxic and nephrotoxic effects [31]

Although studies have been conducted on chronic cya-nide intoxication in various animal species little is knownabout its potentially toxic effects on development Fetalmalformations have been linked to maternal consumptionof cyanogenic plants in animals such as pigs horses sheepand cattle [32ndash36] as well as in humans [37 38] Moreoverstudies of goats conducted in our lab have shown thatmaternal cyanide exposure is associated with fetal abortionand retrognathia [39] We verified these findings in studieswith rats where pups from dams treated with different dosesof cyanide andor thiocyanate (the activemetabolite resultingfrom the biotransformation of cyanide) exhibited similartoxic effects some of which were observed only at weaning[40]

We have previously demonstrated that the pig is a usefulanimal model for assessing chronic cyanide toxicity [31]and others have found it suitable for reproductive toxicologystudies [41 42] The aim of this study was to evaluate thetoxic effects in both dams and their offspring of maternalexposure to potassium cyanide (KCN) during pregnancy inpigs

2 Materials and Methods

This study was conducted at the University of Sao Paulo(USP) Experimental Station Pirassununga SP Brazil(S21∘581015840 W47∘271015840) The procedures were approved by theUSP Animal Ethics Committee and all animal care and han-dling was performed by experienced personnel under veter-inary supervision

21 Animals Feeding and Experimental Design Landrace-Large White sows 240 days old were bred to one male pigof the same breed All females were negative for antibodies toAujeszkyrsquos disease and for infections with Campylobacter spMycobacterium avium Brucella suis and Leptospira sp

The day of breeding was defined as gestational day 1(GD1) and pregnancies were confirmed by ultrasound (US)apparatus (Scanner 100 Vet Pie Medical) on GD21 Afterthis confirmation females began to receive the experimentalrations Pregnant sows were randomly allocated into fourtreatment group (119873 = 6 per group at the beginning of theexperiment) and administered varying doses of KCN (MerckGermany) in mg per kg of body weight (mgkg BW) asfollows 0 (KCN0 group) 20 (KCN2 group) 40 (KCN4group) and 60 (KCN6 group)

The purpose of this study was to assess for embryotoxiceffects of cyanide using swine as amodel organismGiven thatembryo implantation in swine starts on day 13 after breedingand ends on day 18 [43] KCN was administered from GD21to parturition (approximately day 114 of pregnancy) twice aday with one half dose administered between 700 and 0730and the other half dose administered between 1730 and 1800mixed with 10 g of starch biscuits in individual troughs Thecontrol group received only the biscuitsWater and food werefreely available During gestation the dams were weighedweekly

22 Biochemical and Hormonal Evaluations Blood sampleswere collected via jugular venipuncture using heparinizedsyringes at the 21st 50th 80th and 110th days of pregnancyBlood serum was frozen and stored at minus10∘C until analysisCommercial kits (CELM Brazil) were used for the determi-nation of albumin glucose urea creatinine aspartate amino-transferase (AST) and gamma glutamyltranspeptidase(GGT) levels using automatic biochemical system (SBA-200CELM) The plasmatic concentrations of thyroxine (T

4)

and triiodothyronine (T3) were measured on GD110 using

commercial radioimmunoassay kits (Coat-A-Count Siem-ens)

23 Ultrasonographic Evaluation and Pathological Study inSows Ultrasonographic evaluationwas performed onGD45GD55 and GD65 in each pregnant swine to measure the fol-lowing fetal parameters crown-rump length (CRL) thoracicdiameter (TD) abdominal diameter (AD) and biparietaldiameter (BPD)

On GD110 one pregnant sow from each group was euth-anized the amniotic fluid was collected for chemical analysisFragments of the CNS thyroid glands lungs pancreasliver and kidneys were collected and fixed in 10 bufferedformalin embedded in paraffin cut into 5120583m sections andstained by hematoxylin and eosin (HE)

24 Evaluations in Piglets Immediately at birth each neo-natersquos body weight and gender were recorded Each newbornwas then examined carefully for gross abnormalities [44]Two newborn pigs a male and female from each motherwere then euthanized to collect fragments of the CNSthyroid glands lungs pancreas liver and kidneys Frombirth(PND1) to three months of age (PND120) each piglet wasweighed weekly and the weight gains were calculated Theabsolute weights andweight gains of the piglets were analyzed

Journal of Toxicology 3

separately by gender Blood samples from each piglet weretaken via jugular venipuncture immediately after birth and onthe 7th 14th 21st and 45th days of life for the evaluation ofthe same biochemical parameters assessed in their mothers

At the end of the experimental period (PND120) thepiglets were euthanized Fragments of the CNS thyroidpancreas liver and kidney were collected and fixed in 10buffered formalin embedded in paraffin cut into 5 120583msections and stained by hematoxylin and eosin (HE)

25 Analytical Procedures Thiocyanate levels of gilts weredosed in serum at GD21 GD50 GD80 and GD110 in amni-otic fluid on GD110 and into colostrum immediately afterparturition by spectrophotometry using the method outlinedby Pettigrew and Fell [15] with modifications specific to ourstudy Briefly 02mL of the biologicalmaterials were added to18mL of a 10 trichloroacetic acid solution and centrifugedat 10000 rpm for 20minNext 05mLof supernatant aliquotswas acidified with 025mL of 1M HCl The samples weremixed after adding each of the following reagents 50 120583L ofsaturated bromine water 100 120583L of trioxide arsenious (20 gLin 01 NNaOH) 09mLof pyridine (10mLof 12NHCl 60mLof pyridine and 40mL of deionized water) and p-PDAreagent (2 g in 1 L of 05M HCl 3 1 prepared immediatelybefore use) The reddish-pink complex that formed was readat 540 nm after 15min in a spectrophotometer (MicronalB382 Micronal SA Brazil) against a blank sample Thio-cyanate levels in the samples were read based on standardcurves prepared with thiocyanate of known concentrations(25ndash300 120583molL) Data were expressed as 120583LmolL

The linearity of the analytical procedure was tested atconcentrations of 25 50 75 100 200 and 300120583molLof thiocyanate in 10 samples for each concentration Thecoefficient of determination was 09911 slope 00008097and the 119910-axis intercept in 0005267 The intralaboratoryprecision expressed by the coefficient of variation intra- andinterday was obtained by analyzing the levels of thiocyanatein standard samples of 100120583molL in water and pool samplesof serum colostrum and amniotic fluid processed fivetimes each on two different days The coefficient of variationranged from 13 to 58 intra- and 21 to 48 interday Thethiocyanate recovery was from 955 to 983 for serum 945 to966 for colostrum and 988 to 982 for amniotic fluid addedto the standard solution of thiocyanate at concentrations of100 and 200120583molL respectively

26 Statistical Analysis Weight gain data serum biochem-istry ultrasound measurements and serum thiocyanate lev-els were analyzed using a mixed linear model (Proc Mixed)for each treatment The animals were nested within thetreatments and repeated measurements of the variables weretaken over time The animals were considered a randomfactor in the model

Hormone serum levels birth weights and thiocyanatelevels in the colostrumwere analyzed statistically by one-wayanalysis of variance (ANOVA) followed byDunnettrsquos testThefrequency of dead piglets was analyzed using Fisherrsquos exacttest with each treatment compared to the controls

Data are reported as the means plusmn SEM and were analyzedusing SAS software (Version 92 SAS Institute Cary NC) Inall cases the probability of significant differences was set at120572 = 005

Calibration and concentration curves of thiocyanate inthe different biological fluids versus the time taken aftertreatments were made using the GraphPad Prism 500(Graphpad 2007)

3 Results

Pregnant sows did not show clinical signs of acute cyanideintoxication during the experimental period All the damssurvived the treatment throughout the study There wasno significant difference between controls and KCN-treatedsows in body weight gain (Table 1 119875 gt 005)

Fetal deaths were diagnosed by ultrasonographic eval-uation in three females from the KCN4 group and in onepregnant female from KCN6 group between 21 and 45 daysof gestation Females that failed pregnancy were withdrawnfrom the experiment and replaced with pregnant females

Biochemical analysis of serum collected during preg-nancy showed fluctuations of multiple parameters but theseparameters remained within the normal ranges for all swine(data not shown) Similarly no changes in T

3(control 0039 plusmn

0005 KCN2 0033 plusmn 0001 KCN4 0034 plusmn 0002 andKCN6 0034 plusmn 0002) and T

4(control 084 plusmn 04 KCN2

09 plusmn 03 KCN4 079 plusmn 01 and KCN6 093 plusmn 02) levelswere found on the 110th day of gestation On the otherhand plasma thiocyanate levels were increased on GD50GD80 and GD110 in females from all treated groups (Fig-ure 1 119875 lt 005) Colostrum concentrations of thiocyanatewere increased in all dams treated with cyanide (Figure 1119875 lt 005) Amniotic fluid evaluated from one female ineach group showed increased thiocyanate levels in a dose-response fashion (KCN0 205120583molL KCN2 511 120583molLKCN4 559 120583molL and KCN6 657 120583molL)

Ultrasonographic evaluation at GD45 GD55 and GD65demonstrated no significant changes in the morphometricmeasurements (CRL TDAD andBPD) of the fetuses in eachgroup (data not shown)

In histopathological assessments of euthanized pregnantsows at GD110 a greater number of Purkinje cells withacidophilic cytoplasm were found in all experimental sowswhereas vacuolar degeneration of Purkinje cells was observedonly in sows from the KCN6 group (Figure 2) Compared tothe control sows the experimental sows had thyroid follicleenlargement thickening of the cubic follicular epitheliumand reabsorption vacuoles in colloid (Figure 3) Additionallythere was vacuolar degeneration of the renal tubular epithe-lium in sows from all groups exposed to KCN (Figure 4)

There was a significant increase (119875 = 0002) in the num-ber of dead piglets in the KCN4 group compared with theKCN0 group (Table 1) The body weight of the piglets at birthwas unaffected (Table 1 119875 gt 005) by the treatment

The biochemical analysis of serum collected from pigletsshowed fluctuations of the different parameters but theseparameters remained within the normal ranges for swine

4 Journal of Toxicology

Table 1 Reproductive parameters from sows treated with KCN from day 21 of pregnancy to term and the body weight of their piglets at birthuntil 120 days old

Parameters Control (119899 = 6) KCN (mgkg per day)20 (119899 = 6) 40 (119899 = 6) 60 (119899 = 6)

Body weight gain (kg) during pregnancya 696 plusmn 46 552 plusmn 51 498 plusmn 41 594 plusmn 82Number of assisted births 0 (5)b 0 (5) 4 (5) 1 (5)Live piglets 57 55 36 51

Male 27 30 13 24Female 30 25 23 27

Number of dead piglets 6 4 23lowast 10

Stillbirths 5 4 14 7Mummified fetuses 1 0 9 3

Subsequent total born piglets (mean) 126 118 72 94Piglets body weight at birtha 168 plusmn 004 (57)c 164 plusmn 003 (55) 171 plusmn 007 (36) 160 plusmn 005 (51)Overall weight gain (kg) of piglets at 120 days

Males 5525 plusmn 382 5247 plusmn 243 5407 plusmn 297 5279 plusmn 349Females 5382 plusmn 324 5235 plusmn 278 5162 plusmn 197 5162 plusmn 299

aMean plusmn SEMbNumber of deliveriescNumber of piglets weighedlowast

119875 lt 005 compared with controls

0

50

100

150

200

250

KCN0KCN2

KCN4KCN6

21 50 80 110Days of pregnancy

Thio

cyan

ate (

120583m

olL

)

(a)

KCN0KCN2

KCN4KCN6

0

10

20

30

40

50

Colostrum

Thio

cyan

ate (

120583m

olL

)

(b)

Figure 1 Thiocyanate levels (means plusmn SEM) in plasma (a) and colostrum (b) of sows allocated into four treatment groups KCN0 KCN2KCN4 andKCN6 that received respectively 00 20 40 and 60mgkg of bodyweight of cyanide fromday 21 of pregnancy to term 119901 lt 005compared to control

(data not shown) At PND120 there was no treatment effecton the overall weight gain of male or female piglets (Table 1)No histopathological lesions were observed in euthanizednewborns At PND120 piglets in all experimental groups hadsimilar histopathological changes as their mothers includingthyroid follicular enlargement and epithelial thickening andvacuolization (Figure 3)

4 Discussion

The administration of KCN orally was chosen aiming tomimic the main route of natural exposure to this speciesThedoses of KCN used here were chosen based on data from anearlier study conducted in our laboratory on male growing-finishing swine [31] inwhich the animals showed toxic effects

Journal of Toxicology 5

(b) (c)

(a)

Figure 2 Light photomicrograph of pregnant sows CNS (a) animal from control group ((b) and (c) resp) animals treated with 40 and60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing Purkinje cells with acidophilic cytoplasm (arrow (b)) andvacuolar degeneration of Purkinje cells (arrow (c)) Magnification times20 HE

(a) (b)

(c) (d)

Figure 3 Light photomicrograph of pregnant sows thyroid ((a) and (c)) animals from control group ((b) and (d)) animals treated with60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing enlargement of thyroid follicles (b) thickening of thefollicular epithelium (arrow (d)) and reabsorption vacuoles in colloid (arrowhead (d)) Magnification times4 ((a) and (b)) times20 ((c) and (d))HE

of chronic exposure to cyanide but did not manifest anyclinical signs of acute toxicity (eg dyspnea and convulsiveseizures) Similarly none of the pregnant females in thisstudy showed clinical symptoms of acute cyanide intoxicationduring the period of KCN administration

Given that the bitter taste of cyanide could compromisefood consumption during the study [45] starch biscuitsknown to be highly palatable to swine were chosen toborne the cyanide This procedure ensured that all femalesingested the target doses of cyanide quickly (usually in lessthan 2min) in each administration In a previous study oftoxicokinetics in pregnant sows we verified that following

oral exposure to KCN serum thiocyanate levels increase by3 hrs peak after 6 hrs and remain elevated for up to 21 hrs[46] Thus we assumed that the administration of cyanidetwice daily would ensure high serum levels of thiocyanatesteadily independently of the phase of pregnancy

Although some studies have found an associationbetween the reduction of body weight and prolonged expo-sure to cyanide in humans and animals [47ndash49] we did notdetect a decrease in this parameter in any of the pregnant sowsexposed to KCN This result corresponds with our previouswork conducted in growing-finishing pigs [31] Likewisestudies evaluating cyanide exposure during pregnancy in

6 Journal of Toxicology

(a)

(b) (c)

Figure 4 Light photomicrograph of pregnant sows kidney (a) animal from control group ((b) and (c) resp) animals treated with 20 and60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing vacuolar degeneration of the renal tubular epithelium(arrow (b) and (c)) Magnification times20 HE

goats [39] and rats [40] have not found differences in weightgain between the control and experimental animal groups Inthe same manner Tewe and Maner [50] did not find weightchanges in pregnant pigs fed cassava containing high levels ofcyanide

Although no biochemical changes were observed in preg-nant females exposed to KCN the histopathology performedin these sows revealed kidney and liver damage Lesionssimilar to those observed in our study have previously beendescribed in studies of other species chronically exposed tocyanide [30 51 52] as well as in pigs [31] It remains tobe determined if these lesions are a consequence of toxicitydirectly from cyanide thiocyanate or both

Studies clearly show that the CNS is an important targetin prolonged exposure to cyanide in both humans [53] andanimals [54ndash56] In humans it has been described to resultin several neuropathies such as spastic paraparesis (alsocalled ldquokonzordquo) Parkinsonrsquos disease cognitive impairmentand ocular pathologies [11 13 14 16 24 57]

The pig is an emerging model system for studying thehuman CNS [58] In a previous study in our lab where cya-nide was administered to growing pigs during the sameperiod evaluated here we found Purkinje cell degenerationand loss of cerebellar white matter in all groups treatedwith KCN [31] Similar lesions were found in this studyin sows exposed to cyanide These findings strengthen ourassertion that pigs should be used as an animal model tobetter understand the mechanism(s) of neurotoxicity fromcyanide andor its main metabolite

Tropical pancreatic diabetes has been associated withchronic cyanide exposure through consumption of cassavain man [25] and was reproduced in studies of dogs fedcassava [59] However studies administering cyanide todifferent animal species [29 60] including pigs [31] were

not able to reproduce these diabetogenic effects despite theanimals having islet cells functionality similar to humans[61] Given that pregnancy itself has a diabetogenic effectdue to a decrease in insulin sensitivity of maternal tissuesand resultant increase in insulin demand [62 63] pregnantsows would theoretically be evenmore susceptible to developdiabetes upon cyanide exposure Thus we hypothesized thatour study would have offered all the conditions needed toreproduce tropical pancreatic diabetes in pigs However wedid not find any changes in the parameters chosen to evaluatethe diabetogenic effects of cyanide (ie glucose levels andhistopathology) As we have previously shown that pregnantrats exposed to cyanide have increased levels of glucose andpancreatic islet degeneration [40] pregnant rats may be abetter animal model to assess the pancreotoxic effects ofcyanide

Studies have found a correlation between low doses ofchronic cyanide intake with a higher incidence of abortion inmares [32] sheep [36] and goats [39] Chronic cyanide intakein low doses from tobacco smoke has similarly been associ-ated with spontaneous abortion in women [64] although itremains unclear if this finding is specifically due to cyaniderather than other toxic compounds found in cigarettes

Higher rates of fetal death stillbirth assisted birth andmummified piglets were found in sows exposed to cyanidein this study The most common infectious causes of repro-ductive disease in Brazil (Campylobacter sp Mycobacteriumavium Brucella suis and Leptospira sp) were ruled out inthese animals Many causes could be posited to explain thesefinding but it is plausible to consider KCN as the mostlikely one We therefore suggest that the pig is a good animalmodel system for studying the role of cyanide andor its mainmetabolite in smoking-related abortions in women In ourstudy as we did not detect malformations in newborn pigs

Journal of Toxicology 7

it was not possible to conclude if ultrasound evaluation inpigs could be valuable for early detection of malformationsldquoin uterordquo

Amniocenteses in this study revealed high levels ofthiocyanate in the amniotic fluid of pregnant sows Denckeret al [65] and Schwartz [66] show that anionic substancessuch as the weak acid cyanuric acid can accumulate in theembryo and fetus This may be due to the pH gradient inmaternal plasma which is more acidic than the pH of fetalplasma Studies have thus observed an ldquoimprisonmentrdquo ofcyanide in the fetal organism [67] In addition it is knownthat the enzyme rhodanese is found in the placenta and thefetus [68] Thus it is possible that the ldquoarrestedrdquo cyanidecan be transformed into its main metabolite resulting inindirect thiocyanate toxicity to the fetus This hypothesisis additionally supported by a study in which cyanide orthiocyanate was administered to pregnant rats and bothsubstances were found to promote toxic effects in the fetuses[40]

The protocols that have been used to assess develop-mental toxicity in previous studies have certain limitations[69] One such limitation is the lack of postnatal monitoringof the development of the offspring which can result inerroneous conclusions about the developmental toxicity ofthe substances under examination In fact de Sousa et al [40]evaluated prenatal exposure to cyanide in rats and found thatalterations in pups were detected only at weaning Our studysupports these findings as we identified certain toxic effectsof cyanide in the piglets only at weaning

5 Conclusion

In conclusion pregnant sows exposed to cyanide had toxiceffects in both mothers and piglets However the toxiceffects in the offspring were observed belatedly Thereforethe data presented here reinforces the hypothesis that acomplementary evaluation of the neonate after weaning is anecessary component of studies on developmental toxicityIn addition we suggest that swine can serve as a usefulanimal model to study the neurological goitrogenic andreproductive toxicity of cyanide

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] W C Beamer R M Shealy and D S Prough ldquoAcute cyanidepoisoning from laetrile ingestionrdquo Annals of Emergency Medi-cine vol 12 no 7 pp 449ndash451 1983

[2] R Froldi M Cingolani and C Cacaci ldquoA case of suicide byingestion of sodium nitroprussiderdquo Journal of Forensic Sciencesvol 46 no 6 pp 1504ndash1506 2001

[3] J E Poulton ldquoCyanogenic compounds in plants and their toxiceffectsrdquo inHandbook of Natural Toxins Plant and Fungal ToxinsR F Keeler and A R Tu Eds vol 1 pp 117ndash157 Marcel DekkerNew York NY USA 1983

[4] United States Army Medical Research Institute of ChemicalDefense and Chemical Casualty Care Division ldquoCyaniderdquo inMedicalManagement of Chemical Causalities Handbook UnitedStates Army Medical Research Institute of Chemical DefenseAberdeen Md USA 3rd edition 2004

[5] M J Koschel ldquoWhere therersquos smoke there may be cyaniderdquoTheAmerican Journal of Nursing vol 102 no 8 pp 39ndash42 2002

[6] K Anseeuw N Delvau G Burillo-Putze et al ldquoCyanide poi-soning by fire smoke inhalation a European expert consensusrdquoEuropean Journal of Emergency Medicine vol 20 no 1 pp 2ndash92013

[7] V Schulz ldquoClinical pharmacokinetics- of nitroprusside cya-nide thiosulphate and thiocyanaterdquo Clinical Pharmacokineticsvol 9 no 3 pp 239ndash251 1984

[8] J Vetter ldquoPlant cyanogenic glycosidesrdquo Toxicon vol 38 no 1pp 11ndash36 2000

[9] B Ballantyne ldquoToxicology of cyanidesrdquo in Clinical and Exper-imental Toxicology of Cyanides B Ballantyne and T C MarrsEds pp 41ndash126 Wright Bristol UK 1987

[10] H Nzwalo and J Cliff ldquoKonzo from poverty cassava andcyanogen intake to toxico-nutritional neurological diseaserdquoPLoS Neglected Tropical Diseases vol 5 no 6 Article ID e10512011

[11] B O Osuntokun ldquoCassava diet chronic cyanide intoxicationand neuropathy in the Nigerian Africansrdquo World Review ofNutrition and Dietetics vol 36 pp 141ndash173 1981

[12] J Njoh ldquoTropical ataxic neuropathy in Liberiansrdquo Tropical andGeographical Medicine vol 42 no 1 pp 92ndash94 1990

[13] N L Rosenberg J A Myers and W R Wayne Martin ldquoCya-nide-induced parkinsonism clinical MRI and 6-fluorodopaPET studiesrdquo Neurology vol 39 no 1 pp 142ndash144 1989

[14] M Di Filippo N Tambasco G Muzi et al ldquoParkinsonism andcognitive impairment following chronic exposure to potassiumcyaniderdquoMovement Disorders vol 23 no 3 pp 468ndash470 2008

[15] A R Pettigrew and G S Fell ldquoSimplified colorimetric determi-nation of thiocyanate in biological fluids and its application toinvestigation of the toxic amblyopiasrdquo Clinical Chemistry vol18 no 9 pp 996ndash1000 1972

[16] Y Solberg M Rosner and M Belkin ldquoThe association betwencigarette smoking and ocular diseasesrdquo Survey of Ophthalmol-ogy vol 42 no 6 pp 535ndash547 1998

[17] A G Freeman ldquoOptic neuropathy and chronic cyanide intox-ication a reviewrdquo Journal of the Royal Society of Medicine vol81 no 2 pp 103ndash106 1988

[18] I G Syme J Bronte-Stewart and W S Foulds ldquoClinical andbiochemical findings in Leberrsquos hereditary optic atrophyrdquoTrans-actions of the Ophthalmological Societies of the United Kingdomvol 103 no 5 pp 556ndash559 1983

[19] K Tsao P A Aitken and D R Johns ldquoSmoking as an aetio-logical factor in a pedigree with Leberrsquos hereditary optic neu-ropathyrdquo British Journal of Ophthalmology vol 83 no 5 pp577ndash581 1999

[20] S J Crews ldquoBilateral amblyopia in west Indiansrdquo Transactionsof the Ophthalmological Societies of the United Kingdom vol 83pp 653ndash667 1963

[21] A D Mackenzie and C I Phillips ldquoWest Indian amblyopiardquoBrain vol 91 no 2 pp 249ndash260 1968

[22] F D Carroll ldquoJamaican optic neuropathy in immigrants to theUnited StatesrdquoThe American Journal of Ophthalmology vol 71no 1 pp 261ndash265 1971

8 Journal of Toxicology

[23] B O Osuntokun and O Osuntokun ldquoTropical amblyopia inNigeriansrdquo American Journal of Ophthalmology vol 72 no 4pp 708ndash716 1971

[24] K Tucker and T R Hedges ldquoFood shortages and an epidemicof optic and peripheral neuropathy in CubardquoNutrition Reviewsvol 51 no 12 pp 349ndash357 1993

[25] D E McMillan and P J Geevarghese ldquoDietary cyanide andtropical malnutrition diabetesrdquo Diabetes Care vol 2 no 2 pp202ndash208 1979

[26] C S Pitchumoni N K Jain A B Lowenfels and E P DimagnoldquoChronic cyanide poisoning unifying concept for alcoholic andtropical pancreatitisrdquo Pancreas vol 3 no 2 pp 220ndash222 1988

[27] B P Kamalu ldquoPathological changes in growing dogs fed ona balanced cassava (Manihot esculenta Crantz) dietrdquo BritishJournal of Nutrition vol 69 no 3 pp 921ndash934 1993

[28] B P Kamalu ldquoThe adverse effects of long-term cassava (Mani-hot esculenta Crantz) consumptionrdquo International Journal ofFood Sciences and Nutrition vol 46 no 1 pp 65ndash93 1995

[29] B Soto-Blanco S L Gorniak and E T Kimura ldquoPhysiopatho-logical effects of the administration of chronic cyanide togrowing goatsmdasha model for ingestion of cyanogenic plantsrdquoVeterinary ResearchCommunications vol 25 no 5 pp 379ndash3892001

[30] A B Sousa B Soto-Blanco J L Guerra E T Kimura and SL Gorniak ldquoDoes prolonged oral exposure to cyanide promotehepatotoxicity and nephrotoxicityrdquo Toxicology vol 174 no 2pp 87ndash95 2002

[31] H Manzano A B de Sousa B Soto-Blanco J L Guerra P CMaiorka and S L Gorniak ldquoEffects of long-term cyanide inges-tion by pigsrdquo Veterinary Research Communications vol 31 no1 pp 93ndash104 2007

[32] J T Prichard and J L Voss ldquoFetal ankylosis in horses associatedwith hybrid Sudan pasturerdquo Journal of the American VeterinaryMedical Association vol 150 no 8 pp 871ndash873 1967

[33] L A Selby R W Menges E C Houser R E Flatt and A ACase ldquoOutbreak of swine malformations associated with thewild black cherry Prunus serotinardquo Archives of EnvironmentalHealth vol 22 no 4 pp 496ndash501 1971

[34] M G W Rodel ldquoEffects of different grasses on the incidence ofneonatal goiter and skeletal deformities in autumn born lambsrdquoRhodesia Agricultural Journal vol 69 pp 59ndash60 1972

[35] J T Seaman M G Smeal and J C Wright ldquoThe possibleassociation of a sorghum (Sorghum sudanese) hybrid as acause of developmental defects in calvesrdquo Australian VeterinaryJournal vol 57 no 7 pp 351ndash352 1981

[36] G A Bradley H C Metcalf C Reggiardo et al ldquoNeuroaxonaldegeneration in sheep grazing Sorghum pasturesrdquo Journal ofVeterinary Diagnostic Investigation vol 7 no 2 pp 229ndash2361995

[37] J D Singh ldquoThe teratogenic effects of dietary cassava on thepregnant albino rat a preliminary reportrdquo Teratology vol 24no 3 pp 289ndash291 1981

[38] H K Kumbnani A Singh G Singh and P Parimoo ldquoLimbrsquosmalformations in SouthernNigeriamdashGarri (Manihot esculenta)a probable causerdquo Journal of Human Ecology vol 1 pp 189ndash1911990

[39] B Soto-Blanco and S L Gorniak ldquoPrenatal toxicity of cyanidein goatsmdasha model for teratological studies in ruminantsrdquoTheriogenology vol 62 no 6 pp 1012ndash1026 2004

[40] A B de Sousa P CMaiorka I DGoncalves L RM de Sa andS L Gorniak ldquoEvaluation of effects of prenatal exposure to the

cyanide and thiocyanate inwistar ratsrdquoReproductive Toxicologyvol 23 no 4 pp 568ndash577 2007

[41] N C Ganderup W Harvey J T Mortensen and W HarroukldquoTheminipig as nonrodent species in toxicologymdashwhere are wenowrdquo International Journal of Toxicology vol 31 no 6 pp 507ndash528 2012

[42] G Bode P Clausing F Gervais et al ldquoThe utility of theminipigas an animal model in regulatory toxicologyrdquo Journal of Phar-macological and Toxicological Methods vol 62 no 3 pp 196ndash220 2010

[43] R D Geisert R H Renegar W W Thatcher R M Robertsand FW Bazer ldquoEstablishment of pregnancy in the pig I Inter-relationships between preimplantation development of the pigblastocyst and uterine endometrial secretionsrdquoBiology of Repro-duction vol 27 no 4 pp 925ndash939 1982

[44] K T Szabo Congenital Malformations in Laboratory and FarmAnimals Academic Press San Diego Calif USA 1989

[45] E A Iyayi ldquoDietary cyanide effect on performance and serumtestosterone of growing male pigsrdquo Beitrage zur TropischenLandwirtschaft undVeterinarmedizin vol 29 no 3 pp 347ndash3521991

[46] H Manzano A B Souza and S L Gorniak ldquoExposicaocianıdrica em suınos uma abordagem dos parametros toxic-ocineticos utilizando o tiocianato como biomarcadorrdquoBrazilianJournal of Veterinary Research and Animal Science vol 43 pp93ndash101 2006

[47] P Blanc M Hogan K Mallin D Hryhorczuk S Hessl and BBernard ldquoCyanide intoxication among silver-reclaiming work-ersrdquo The Journal of the American Medical Association vol 253no 3 pp 367ndash371 1985

[48] C F I Onwuka ldquoHydrocyanic acid contents of tropical browseand their influence on performance of goatsrdquo Food Chemistryvol 45 no 1 pp 5ndash10 1992

[49] C Ibebunjo B P Kamalu and E C Ihemelandu ldquoComparisonof the effects of cassava (Manihot esculenta Crantz) organiccyanide and inorganic cyanide on muscle and bone develop-ment in a Nigerian breed of dogrdquo British Journal of Nutritionvol 68 no 2 pp 483ndash491 1992

[50] O O Tewe and J H Maner ldquoPerformance and pathophysio-logical changes in pregnant pigs fed cassava diets containingdifferent levels of cyaniderdquo Research in Veterinary Science vol30 no 2 pp 147ndash151 1981

[51] N P Okolie and A U Osagie ldquoLiver and kidney lesions andassociated enzyme changes induced in rabbits by chronic cya-nide exposurerdquo Food and Chemical Toxicology vol 37 no 7 pp745ndash750 1999

[52] B Soto-Blanco and S L Gorniak ldquoToxic effects of prolongedadministration of leaves of cassava (Manihot esculenta Crantz)to goatsrdquo Experimental and Toxicologic Pathology vol 62 no 4pp 361ndash366 2010

[53] J Wilson ldquoCyanide in human disease a review of clinical andlaboratory evidencerdquo Fundamental and Applied Toxicology vol3 no 5 pp 397ndash399 1983

[54] E M Mills P G Gunasekar L Li J L Borowitz and G EIsom ldquoDifferential susceptibility of brain areas cyanide involvesdifferent modes of cell deathrdquo Toxicology and Applied Pharma-cology vol 156 no 1 pp 6ndash16 1999

[55] B Soto-Blanco P CesarMarioka and S Lima Gorniak ldquoEffectsof long-term low-dose cyanide administration to ratsrdquo Ecotoxi-cology and Environmental Safety vol 53 no 1 pp 37ndash41 2002

Journal of Toxicology 9

[56] B Soto-Blanco F T V Pereira A F D CarvalhoM AMiglinoand S L Gorniak ldquoFetal andmaternal lesions of cyanide dosingto pregnant goatsrdquo Small Ruminant Research vol 87 no 1ndash3 pp76ndash80 2009

[57] T Tylleskar M Banea N Bikangi G Nahimana L-A Perssonand H Rosling ldquoDietary determinants of a non-progressivespastic paraparesis (Konzo) a case-referent study in a highincidence area of Zairerdquo International Journal of Epidemiologyvol 24 no 5 pp 949ndash956 1995

[58] M M Swindle A Makin A J Herron F J Clubb Jr and K SFrazier ldquoSwine asmodels in biomedical research and toxicologytestingrdquo Veterinary Pathology vol 49 no 2 pp 344ndash356 2012

[59] B P Kamalu ldquoThe effect of a nutritionally-balanced cassava(Manihot esculenta Crantz) diet on endocrine function usingthe dog as a model 1 Pancreasrdquo British Journal of Nutrition vol65 no 3 pp 365ndash372 1991

[60] N P Okolie and A U Osagie ldquoDifferential effects of chroniccyanide intoxication on heart lung and pancreatic tissuesrdquo Foodand Chemical Toxicology vol 38 no 6 pp 543ndash548 2000

[61] M M Swindle and A C Smith ldquoComparative anatomy andphysiology of the pigrdquo Scandinavian Journal of LaboratoryAnimal Science vol 25 no 1 pp 11ndash21 1998

[62] L Jovanovic andD J Pettitt ldquoGestational diabetesmellitusrdquoTheJournal of the AmericanMedical Association vol 286 no 20 pp2516ndash2518 2001

[63] TA Buchanan andAHXiang ldquoGestational diabetesmellitusrdquoThe Journal of Clinical Investigation vol 115 no 3 pp 485ndash4912005

[64] The Practice Committee of American Society for ReproductiveMedicine ldquoSmoking and infertilityrdquo Fertility and Sterility vol4 pp 1181ndash1186 2004

[65] L Dencker R DrsquoArgy B R G Danielsson H Ghantous and GO Sperber ldquoSaturable accumulation of retinoic acid in neuraland neural crest derived cells in early embryonic developmentrdquoDevelopmental Pharmacology andTherapeutics vol 10 no 3 pp212ndash223 1987

[66] S Schwartz ldquoProviding toxicokinetic support for reproductivetoxicology studies in pharmaceutical developmentrdquo Archives ofToxicology vol 75 no 7 pp 381ndash387 2001

[67] W J Scott and H Nau ldquoAccumulation of weak acids in theyoung mammalian embryordquo in Pharmacokinetics in Teratoge-nesis H Nau and W J Scott Eds pp 72ndash76 CRC Press BocaRaton Fla USA 1987

[68] O O Tewe J H Maner and G Gomez ldquoInfluence of cassavadiets on placental thiocyanate transfer tissue rhodanese activityand performance of rats during gestationrdquo Journal of the Scienceof Food and Agriculture vol 28 no 8 pp 750ndash756 1977

[69] L Claudio C F Bearer andDWallinga ldquoAssessment of theUSenvironmental protection agency methods for identification ofhazards to developing organisms part II the developing toxicitytesting guidelinerdquo American Journal of Industrial Medicine vol35 no 6 pp 554ndash563 1999

Journal of Toxicology 3

separately by gender Blood samples from each piglet weretaken via jugular venipuncture immediately after birth and onthe 7th 14th 21st and 45th days of life for the evaluation ofthe same biochemical parameters assessed in their mothers

At the end of the experimental period (PND120) thepiglets were euthanized Fragments of the CNS thyroidpancreas liver and kidney were collected and fixed in 10buffered formalin embedded in paraffin cut into 5 120583msections and stained by hematoxylin and eosin (HE)

25 Analytical Procedures Thiocyanate levels of gilts weredosed in serum at GD21 GD50 GD80 and GD110 in amni-otic fluid on GD110 and into colostrum immediately afterparturition by spectrophotometry using the method outlinedby Pettigrew and Fell [15] with modifications specific to ourstudy Briefly 02mL of the biologicalmaterials were added to18mL of a 10 trichloroacetic acid solution and centrifugedat 10000 rpm for 20minNext 05mLof supernatant aliquotswas acidified with 025mL of 1M HCl The samples weremixed after adding each of the following reagents 50 120583L ofsaturated bromine water 100 120583L of trioxide arsenious (20 gLin 01 NNaOH) 09mLof pyridine (10mLof 12NHCl 60mLof pyridine and 40mL of deionized water) and p-PDAreagent (2 g in 1 L of 05M HCl 3 1 prepared immediatelybefore use) The reddish-pink complex that formed was readat 540 nm after 15min in a spectrophotometer (MicronalB382 Micronal SA Brazil) against a blank sample Thio-cyanate levels in the samples were read based on standardcurves prepared with thiocyanate of known concentrations(25ndash300 120583molL) Data were expressed as 120583LmolL

The linearity of the analytical procedure was tested atconcentrations of 25 50 75 100 200 and 300120583molLof thiocyanate in 10 samples for each concentration Thecoefficient of determination was 09911 slope 00008097and the 119910-axis intercept in 0005267 The intralaboratoryprecision expressed by the coefficient of variation intra- andinterday was obtained by analyzing the levels of thiocyanatein standard samples of 100120583molL in water and pool samplesof serum colostrum and amniotic fluid processed fivetimes each on two different days The coefficient of variationranged from 13 to 58 intra- and 21 to 48 interday Thethiocyanate recovery was from 955 to 983 for serum 945 to966 for colostrum and 988 to 982 for amniotic fluid addedto the standard solution of thiocyanate at concentrations of100 and 200120583molL respectively

26 Statistical Analysis Weight gain data serum biochem-istry ultrasound measurements and serum thiocyanate lev-els were analyzed using a mixed linear model (Proc Mixed)for each treatment The animals were nested within thetreatments and repeated measurements of the variables weretaken over time The animals were considered a randomfactor in the model

Hormone serum levels birth weights and thiocyanatelevels in the colostrumwere analyzed statistically by one-wayanalysis of variance (ANOVA) followed byDunnettrsquos testThefrequency of dead piglets was analyzed using Fisherrsquos exacttest with each treatment compared to the controls

Data are reported as the means plusmn SEM and were analyzedusing SAS software (Version 92 SAS Institute Cary NC) Inall cases the probability of significant differences was set at120572 = 005

Calibration and concentration curves of thiocyanate inthe different biological fluids versus the time taken aftertreatments were made using the GraphPad Prism 500(Graphpad 2007)

3 Results

Pregnant sows did not show clinical signs of acute cyanideintoxication during the experimental period All the damssurvived the treatment throughout the study There wasno significant difference between controls and KCN-treatedsows in body weight gain (Table 1 119875 gt 005)

Fetal deaths were diagnosed by ultrasonographic eval-uation in three females from the KCN4 group and in onepregnant female from KCN6 group between 21 and 45 daysof gestation Females that failed pregnancy were withdrawnfrom the experiment and replaced with pregnant females

Biochemical analysis of serum collected during preg-nancy showed fluctuations of multiple parameters but theseparameters remained within the normal ranges for all swine(data not shown) Similarly no changes in T

3(control 0039 plusmn

0005 KCN2 0033 plusmn 0001 KCN4 0034 plusmn 0002 andKCN6 0034 plusmn 0002) and T

4(control 084 plusmn 04 KCN2

09 plusmn 03 KCN4 079 plusmn 01 and KCN6 093 plusmn 02) levelswere found on the 110th day of gestation On the otherhand plasma thiocyanate levels were increased on GD50GD80 and GD110 in females from all treated groups (Fig-ure 1 119875 lt 005) Colostrum concentrations of thiocyanatewere increased in all dams treated with cyanide (Figure 1119875 lt 005) Amniotic fluid evaluated from one female ineach group showed increased thiocyanate levels in a dose-response fashion (KCN0 205120583molL KCN2 511 120583molLKCN4 559 120583molL and KCN6 657 120583molL)

Ultrasonographic evaluation at GD45 GD55 and GD65demonstrated no significant changes in the morphometricmeasurements (CRL TDAD andBPD) of the fetuses in eachgroup (data not shown)

In histopathological assessments of euthanized pregnantsows at GD110 a greater number of Purkinje cells withacidophilic cytoplasm were found in all experimental sowswhereas vacuolar degeneration of Purkinje cells was observedonly in sows from the KCN6 group (Figure 2) Compared tothe control sows the experimental sows had thyroid follicleenlargement thickening of the cubic follicular epitheliumand reabsorption vacuoles in colloid (Figure 3) Additionallythere was vacuolar degeneration of the renal tubular epithe-lium in sows from all groups exposed to KCN (Figure 4)

There was a significant increase (119875 = 0002) in the num-ber of dead piglets in the KCN4 group compared with theKCN0 group (Table 1) The body weight of the piglets at birthwas unaffected (Table 1 119875 gt 005) by the treatment

The biochemical analysis of serum collected from pigletsshowed fluctuations of the different parameters but theseparameters remained within the normal ranges for swine

4 Journal of Toxicology

Table 1 Reproductive parameters from sows treated with KCN from day 21 of pregnancy to term and the body weight of their piglets at birthuntil 120 days old

Parameters Control (119899 = 6) KCN (mgkg per day)20 (119899 = 6) 40 (119899 = 6) 60 (119899 = 6)

Body weight gain (kg) during pregnancya 696 plusmn 46 552 plusmn 51 498 plusmn 41 594 plusmn 82Number of assisted births 0 (5)b 0 (5) 4 (5) 1 (5)Live piglets 57 55 36 51

Male 27 30 13 24Female 30 25 23 27

Number of dead piglets 6 4 23lowast 10

Stillbirths 5 4 14 7Mummified fetuses 1 0 9 3

Subsequent total born piglets (mean) 126 118 72 94Piglets body weight at birtha 168 plusmn 004 (57)c 164 plusmn 003 (55) 171 plusmn 007 (36) 160 plusmn 005 (51)Overall weight gain (kg) of piglets at 120 days

Males 5525 plusmn 382 5247 plusmn 243 5407 plusmn 297 5279 plusmn 349Females 5382 plusmn 324 5235 plusmn 278 5162 plusmn 197 5162 plusmn 299

aMean plusmn SEMbNumber of deliveriescNumber of piglets weighedlowast

119875 lt 005 compared with controls

0

50

100

150

200

250

KCN0KCN2

KCN4KCN6

21 50 80 110Days of pregnancy

Thio

cyan

ate (

120583m

olL

)

(a)

KCN0KCN2

KCN4KCN6

0

10

20

30

40

50

Colostrum

Thio

cyan

ate (

120583m

olL

)

(b)

Figure 1 Thiocyanate levels (means plusmn SEM) in plasma (a) and colostrum (b) of sows allocated into four treatment groups KCN0 KCN2KCN4 andKCN6 that received respectively 00 20 40 and 60mgkg of bodyweight of cyanide fromday 21 of pregnancy to term 119901 lt 005compared to control

(data not shown) At PND120 there was no treatment effecton the overall weight gain of male or female piglets (Table 1)No histopathological lesions were observed in euthanizednewborns At PND120 piglets in all experimental groups hadsimilar histopathological changes as their mothers includingthyroid follicular enlargement and epithelial thickening andvacuolization (Figure 3)

4 Discussion

The administration of KCN orally was chosen aiming tomimic the main route of natural exposure to this speciesThedoses of KCN used here were chosen based on data from anearlier study conducted in our laboratory on male growing-finishing swine [31] inwhich the animals showed toxic effects

Journal of Toxicology 5

(b) (c)

(a)

Figure 2 Light photomicrograph of pregnant sows CNS (a) animal from control group ((b) and (c) resp) animals treated with 40 and60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing Purkinje cells with acidophilic cytoplasm (arrow (b)) andvacuolar degeneration of Purkinje cells (arrow (c)) Magnification times20 HE

(a) (b)

(c) (d)

Figure 3 Light photomicrograph of pregnant sows thyroid ((a) and (c)) animals from control group ((b) and (d)) animals treated with60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing enlargement of thyroid follicles (b) thickening of thefollicular epithelium (arrow (d)) and reabsorption vacuoles in colloid (arrowhead (d)) Magnification times4 ((a) and (b)) times20 ((c) and (d))HE

of chronic exposure to cyanide but did not manifest anyclinical signs of acute toxicity (eg dyspnea and convulsiveseizures) Similarly none of the pregnant females in thisstudy showed clinical symptoms of acute cyanide intoxicationduring the period of KCN administration

Given that the bitter taste of cyanide could compromisefood consumption during the study [45] starch biscuitsknown to be highly palatable to swine were chosen toborne the cyanide This procedure ensured that all femalesingested the target doses of cyanide quickly (usually in lessthan 2min) in each administration In a previous study oftoxicokinetics in pregnant sows we verified that following

oral exposure to KCN serum thiocyanate levels increase by3 hrs peak after 6 hrs and remain elevated for up to 21 hrs[46] Thus we assumed that the administration of cyanidetwice daily would ensure high serum levels of thiocyanatesteadily independently of the phase of pregnancy

Although some studies have found an associationbetween the reduction of body weight and prolonged expo-sure to cyanide in humans and animals [47ndash49] we did notdetect a decrease in this parameter in any of the pregnant sowsexposed to KCN This result corresponds with our previouswork conducted in growing-finishing pigs [31] Likewisestudies evaluating cyanide exposure during pregnancy in

6 Journal of Toxicology

(a)

(b) (c)

Figure 4 Light photomicrograph of pregnant sows kidney (a) animal from control group ((b) and (c) resp) animals treated with 20 and60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing vacuolar degeneration of the renal tubular epithelium(arrow (b) and (c)) Magnification times20 HE

goats [39] and rats [40] have not found differences in weightgain between the control and experimental animal groups Inthe same manner Tewe and Maner [50] did not find weightchanges in pregnant pigs fed cassava containing high levels ofcyanide

Although no biochemical changes were observed in preg-nant females exposed to KCN the histopathology performedin these sows revealed kidney and liver damage Lesionssimilar to those observed in our study have previously beendescribed in studies of other species chronically exposed tocyanide [30 51 52] as well as in pigs [31] It remains tobe determined if these lesions are a consequence of toxicitydirectly from cyanide thiocyanate or both

Studies clearly show that the CNS is an important targetin prolonged exposure to cyanide in both humans [53] andanimals [54ndash56] In humans it has been described to resultin several neuropathies such as spastic paraparesis (alsocalled ldquokonzordquo) Parkinsonrsquos disease cognitive impairmentand ocular pathologies [11 13 14 16 24 57]

The pig is an emerging model system for studying thehuman CNS [58] In a previous study in our lab where cya-nide was administered to growing pigs during the sameperiod evaluated here we found Purkinje cell degenerationand loss of cerebellar white matter in all groups treatedwith KCN [31] Similar lesions were found in this studyin sows exposed to cyanide These findings strengthen ourassertion that pigs should be used as an animal model tobetter understand the mechanism(s) of neurotoxicity fromcyanide andor its main metabolite

Tropical pancreatic diabetes has been associated withchronic cyanide exposure through consumption of cassavain man [25] and was reproduced in studies of dogs fedcassava [59] However studies administering cyanide todifferent animal species [29 60] including pigs [31] were

not able to reproduce these diabetogenic effects despite theanimals having islet cells functionality similar to humans[61] Given that pregnancy itself has a diabetogenic effectdue to a decrease in insulin sensitivity of maternal tissuesand resultant increase in insulin demand [62 63] pregnantsows would theoretically be evenmore susceptible to developdiabetes upon cyanide exposure Thus we hypothesized thatour study would have offered all the conditions needed toreproduce tropical pancreatic diabetes in pigs However wedid not find any changes in the parameters chosen to evaluatethe diabetogenic effects of cyanide (ie glucose levels andhistopathology) As we have previously shown that pregnantrats exposed to cyanide have increased levels of glucose andpancreatic islet degeneration [40] pregnant rats may be abetter animal model to assess the pancreotoxic effects ofcyanide

Studies have found a correlation between low doses ofchronic cyanide intake with a higher incidence of abortion inmares [32] sheep [36] and goats [39] Chronic cyanide intakein low doses from tobacco smoke has similarly been associ-ated with spontaneous abortion in women [64] although itremains unclear if this finding is specifically due to cyaniderather than other toxic compounds found in cigarettes

Higher rates of fetal death stillbirth assisted birth andmummified piglets were found in sows exposed to cyanidein this study The most common infectious causes of repro-ductive disease in Brazil (Campylobacter sp Mycobacteriumavium Brucella suis and Leptospira sp) were ruled out inthese animals Many causes could be posited to explain thesefinding but it is plausible to consider KCN as the mostlikely one We therefore suggest that the pig is a good animalmodel system for studying the role of cyanide andor its mainmetabolite in smoking-related abortions in women In ourstudy as we did not detect malformations in newborn pigs

Journal of Toxicology 7

it was not possible to conclude if ultrasound evaluation inpigs could be valuable for early detection of malformationsldquoin uterordquo

Amniocenteses in this study revealed high levels ofthiocyanate in the amniotic fluid of pregnant sows Denckeret al [65] and Schwartz [66] show that anionic substancessuch as the weak acid cyanuric acid can accumulate in theembryo and fetus This may be due to the pH gradient inmaternal plasma which is more acidic than the pH of fetalplasma Studies have thus observed an ldquoimprisonmentrdquo ofcyanide in the fetal organism [67] In addition it is knownthat the enzyme rhodanese is found in the placenta and thefetus [68] Thus it is possible that the ldquoarrestedrdquo cyanidecan be transformed into its main metabolite resulting inindirect thiocyanate toxicity to the fetus This hypothesisis additionally supported by a study in which cyanide orthiocyanate was administered to pregnant rats and bothsubstances were found to promote toxic effects in the fetuses[40]

The protocols that have been used to assess develop-mental toxicity in previous studies have certain limitations[69] One such limitation is the lack of postnatal monitoringof the development of the offspring which can result inerroneous conclusions about the developmental toxicity ofthe substances under examination In fact de Sousa et al [40]evaluated prenatal exposure to cyanide in rats and found thatalterations in pups were detected only at weaning Our studysupports these findings as we identified certain toxic effectsof cyanide in the piglets only at weaning

5 Conclusion

In conclusion pregnant sows exposed to cyanide had toxiceffects in both mothers and piglets However the toxiceffects in the offspring were observed belatedly Thereforethe data presented here reinforces the hypothesis that acomplementary evaluation of the neonate after weaning is anecessary component of studies on developmental toxicityIn addition we suggest that swine can serve as a usefulanimal model to study the neurological goitrogenic andreproductive toxicity of cyanide

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] W C Beamer R M Shealy and D S Prough ldquoAcute cyanidepoisoning from laetrile ingestionrdquo Annals of Emergency Medi-cine vol 12 no 7 pp 449ndash451 1983

[2] R Froldi M Cingolani and C Cacaci ldquoA case of suicide byingestion of sodium nitroprussiderdquo Journal of Forensic Sciencesvol 46 no 6 pp 1504ndash1506 2001

[3] J E Poulton ldquoCyanogenic compounds in plants and their toxiceffectsrdquo inHandbook of Natural Toxins Plant and Fungal ToxinsR F Keeler and A R Tu Eds vol 1 pp 117ndash157 Marcel DekkerNew York NY USA 1983

[4] United States Army Medical Research Institute of ChemicalDefense and Chemical Casualty Care Division ldquoCyaniderdquo inMedicalManagement of Chemical Causalities Handbook UnitedStates Army Medical Research Institute of Chemical DefenseAberdeen Md USA 3rd edition 2004

[5] M J Koschel ldquoWhere therersquos smoke there may be cyaniderdquoTheAmerican Journal of Nursing vol 102 no 8 pp 39ndash42 2002

[6] K Anseeuw N Delvau G Burillo-Putze et al ldquoCyanide poi-soning by fire smoke inhalation a European expert consensusrdquoEuropean Journal of Emergency Medicine vol 20 no 1 pp 2ndash92013

[7] V Schulz ldquoClinical pharmacokinetics- of nitroprusside cya-nide thiosulphate and thiocyanaterdquo Clinical Pharmacokineticsvol 9 no 3 pp 239ndash251 1984

[8] J Vetter ldquoPlant cyanogenic glycosidesrdquo Toxicon vol 38 no 1pp 11ndash36 2000

[9] B Ballantyne ldquoToxicology of cyanidesrdquo in Clinical and Exper-imental Toxicology of Cyanides B Ballantyne and T C MarrsEds pp 41ndash126 Wright Bristol UK 1987

[10] H Nzwalo and J Cliff ldquoKonzo from poverty cassava andcyanogen intake to toxico-nutritional neurological diseaserdquoPLoS Neglected Tropical Diseases vol 5 no 6 Article ID e10512011

[11] B O Osuntokun ldquoCassava diet chronic cyanide intoxicationand neuropathy in the Nigerian Africansrdquo World Review ofNutrition and Dietetics vol 36 pp 141ndash173 1981

[12] J Njoh ldquoTropical ataxic neuropathy in Liberiansrdquo Tropical andGeographical Medicine vol 42 no 1 pp 92ndash94 1990

[13] N L Rosenberg J A Myers and W R Wayne Martin ldquoCya-nide-induced parkinsonism clinical MRI and 6-fluorodopaPET studiesrdquo Neurology vol 39 no 1 pp 142ndash144 1989

[14] M Di Filippo N Tambasco G Muzi et al ldquoParkinsonism andcognitive impairment following chronic exposure to potassiumcyaniderdquoMovement Disorders vol 23 no 3 pp 468ndash470 2008

[15] A R Pettigrew and G S Fell ldquoSimplified colorimetric determi-nation of thiocyanate in biological fluids and its application toinvestigation of the toxic amblyopiasrdquo Clinical Chemistry vol18 no 9 pp 996ndash1000 1972

[16] Y Solberg M Rosner and M Belkin ldquoThe association betwencigarette smoking and ocular diseasesrdquo Survey of Ophthalmol-ogy vol 42 no 6 pp 535ndash547 1998

[17] A G Freeman ldquoOptic neuropathy and chronic cyanide intox-ication a reviewrdquo Journal of the Royal Society of Medicine vol81 no 2 pp 103ndash106 1988

[18] I G Syme J Bronte-Stewart and W S Foulds ldquoClinical andbiochemical findings in Leberrsquos hereditary optic atrophyrdquoTrans-actions of the Ophthalmological Societies of the United Kingdomvol 103 no 5 pp 556ndash559 1983

[19] K Tsao P A Aitken and D R Johns ldquoSmoking as an aetio-logical factor in a pedigree with Leberrsquos hereditary optic neu-ropathyrdquo British Journal of Ophthalmology vol 83 no 5 pp577ndash581 1999

[20] S J Crews ldquoBilateral amblyopia in west Indiansrdquo Transactionsof the Ophthalmological Societies of the United Kingdom vol 83pp 653ndash667 1963

[21] A D Mackenzie and C I Phillips ldquoWest Indian amblyopiardquoBrain vol 91 no 2 pp 249ndash260 1968

[22] F D Carroll ldquoJamaican optic neuropathy in immigrants to theUnited StatesrdquoThe American Journal of Ophthalmology vol 71no 1 pp 261ndash265 1971

8 Journal of Toxicology

[23] B O Osuntokun and O Osuntokun ldquoTropical amblyopia inNigeriansrdquo American Journal of Ophthalmology vol 72 no 4pp 708ndash716 1971

[24] K Tucker and T R Hedges ldquoFood shortages and an epidemicof optic and peripheral neuropathy in CubardquoNutrition Reviewsvol 51 no 12 pp 349ndash357 1993

[25] D E McMillan and P J Geevarghese ldquoDietary cyanide andtropical malnutrition diabetesrdquo Diabetes Care vol 2 no 2 pp202ndash208 1979

[26] C S Pitchumoni N K Jain A B Lowenfels and E P DimagnoldquoChronic cyanide poisoning unifying concept for alcoholic andtropical pancreatitisrdquo Pancreas vol 3 no 2 pp 220ndash222 1988

[27] B P Kamalu ldquoPathological changes in growing dogs fed ona balanced cassava (Manihot esculenta Crantz) dietrdquo BritishJournal of Nutrition vol 69 no 3 pp 921ndash934 1993

[28] B P Kamalu ldquoThe adverse effects of long-term cassava (Mani-hot esculenta Crantz) consumptionrdquo International Journal ofFood Sciences and Nutrition vol 46 no 1 pp 65ndash93 1995

[29] B Soto-Blanco S L Gorniak and E T Kimura ldquoPhysiopatho-logical effects of the administration of chronic cyanide togrowing goatsmdasha model for ingestion of cyanogenic plantsrdquoVeterinary ResearchCommunications vol 25 no 5 pp 379ndash3892001

[30] A B Sousa B Soto-Blanco J L Guerra E T Kimura and SL Gorniak ldquoDoes prolonged oral exposure to cyanide promotehepatotoxicity and nephrotoxicityrdquo Toxicology vol 174 no 2pp 87ndash95 2002

[31] H Manzano A B de Sousa B Soto-Blanco J L Guerra P CMaiorka and S L Gorniak ldquoEffects of long-term cyanide inges-tion by pigsrdquo Veterinary Research Communications vol 31 no1 pp 93ndash104 2007

[32] J T Prichard and J L Voss ldquoFetal ankylosis in horses associatedwith hybrid Sudan pasturerdquo Journal of the American VeterinaryMedical Association vol 150 no 8 pp 871ndash873 1967

[33] L A Selby R W Menges E C Houser R E Flatt and A ACase ldquoOutbreak of swine malformations associated with thewild black cherry Prunus serotinardquo Archives of EnvironmentalHealth vol 22 no 4 pp 496ndash501 1971

[34] M G W Rodel ldquoEffects of different grasses on the incidence ofneonatal goiter and skeletal deformities in autumn born lambsrdquoRhodesia Agricultural Journal vol 69 pp 59ndash60 1972

[35] J T Seaman M G Smeal and J C Wright ldquoThe possibleassociation of a sorghum (Sorghum sudanese) hybrid as acause of developmental defects in calvesrdquo Australian VeterinaryJournal vol 57 no 7 pp 351ndash352 1981

[36] G A Bradley H C Metcalf C Reggiardo et al ldquoNeuroaxonaldegeneration in sheep grazing Sorghum pasturesrdquo Journal ofVeterinary Diagnostic Investigation vol 7 no 2 pp 229ndash2361995

[37] J D Singh ldquoThe teratogenic effects of dietary cassava on thepregnant albino rat a preliminary reportrdquo Teratology vol 24no 3 pp 289ndash291 1981

[38] H K Kumbnani A Singh G Singh and P Parimoo ldquoLimbrsquosmalformations in SouthernNigeriamdashGarri (Manihot esculenta)a probable causerdquo Journal of Human Ecology vol 1 pp 189ndash1911990

[39] B Soto-Blanco and S L Gorniak ldquoPrenatal toxicity of cyanidein goatsmdasha model for teratological studies in ruminantsrdquoTheriogenology vol 62 no 6 pp 1012ndash1026 2004

[40] A B de Sousa P CMaiorka I DGoncalves L RM de Sa andS L Gorniak ldquoEvaluation of effects of prenatal exposure to the

cyanide and thiocyanate inwistar ratsrdquoReproductive Toxicologyvol 23 no 4 pp 568ndash577 2007

[41] N C Ganderup W Harvey J T Mortensen and W HarroukldquoTheminipig as nonrodent species in toxicologymdashwhere are wenowrdquo International Journal of Toxicology vol 31 no 6 pp 507ndash528 2012

[42] G Bode P Clausing F Gervais et al ldquoThe utility of theminipigas an animal model in regulatory toxicologyrdquo Journal of Phar-macological and Toxicological Methods vol 62 no 3 pp 196ndash220 2010

[43] R D Geisert R H Renegar W W Thatcher R M Robertsand FW Bazer ldquoEstablishment of pregnancy in the pig I Inter-relationships between preimplantation development of the pigblastocyst and uterine endometrial secretionsrdquoBiology of Repro-duction vol 27 no 4 pp 925ndash939 1982

[44] K T Szabo Congenital Malformations in Laboratory and FarmAnimals Academic Press San Diego Calif USA 1989

[45] E A Iyayi ldquoDietary cyanide effect on performance and serumtestosterone of growing male pigsrdquo Beitrage zur TropischenLandwirtschaft undVeterinarmedizin vol 29 no 3 pp 347ndash3521991

[46] H Manzano A B Souza and S L Gorniak ldquoExposicaocianıdrica em suınos uma abordagem dos parametros toxic-ocineticos utilizando o tiocianato como biomarcadorrdquoBrazilianJournal of Veterinary Research and Animal Science vol 43 pp93ndash101 2006

[47] P Blanc M Hogan K Mallin D Hryhorczuk S Hessl and BBernard ldquoCyanide intoxication among silver-reclaiming work-ersrdquo The Journal of the American Medical Association vol 253no 3 pp 367ndash371 1985

[48] C F I Onwuka ldquoHydrocyanic acid contents of tropical browseand their influence on performance of goatsrdquo Food Chemistryvol 45 no 1 pp 5ndash10 1992

[49] C Ibebunjo B P Kamalu and E C Ihemelandu ldquoComparisonof the effects of cassava (Manihot esculenta Crantz) organiccyanide and inorganic cyanide on muscle and bone develop-ment in a Nigerian breed of dogrdquo British Journal of Nutritionvol 68 no 2 pp 483ndash491 1992

[50] O O Tewe and J H Maner ldquoPerformance and pathophysio-logical changes in pregnant pigs fed cassava diets containingdifferent levels of cyaniderdquo Research in Veterinary Science vol30 no 2 pp 147ndash151 1981

[51] N P Okolie and A U Osagie ldquoLiver and kidney lesions andassociated enzyme changes induced in rabbits by chronic cya-nide exposurerdquo Food and Chemical Toxicology vol 37 no 7 pp745ndash750 1999

[52] B Soto-Blanco and S L Gorniak ldquoToxic effects of prolongedadministration of leaves of cassava (Manihot esculenta Crantz)to goatsrdquo Experimental and Toxicologic Pathology vol 62 no 4pp 361ndash366 2010

[53] J Wilson ldquoCyanide in human disease a review of clinical andlaboratory evidencerdquo Fundamental and Applied Toxicology vol3 no 5 pp 397ndash399 1983

[54] E M Mills P G Gunasekar L Li J L Borowitz and G EIsom ldquoDifferential susceptibility of brain areas cyanide involvesdifferent modes of cell deathrdquo Toxicology and Applied Pharma-cology vol 156 no 1 pp 6ndash16 1999

[55] B Soto-Blanco P CesarMarioka and S Lima Gorniak ldquoEffectsof long-term low-dose cyanide administration to ratsrdquo Ecotoxi-cology and Environmental Safety vol 53 no 1 pp 37ndash41 2002

Journal of Toxicology 9

[56] B Soto-Blanco F T V Pereira A F D CarvalhoM AMiglinoand S L Gorniak ldquoFetal andmaternal lesions of cyanide dosingto pregnant goatsrdquo Small Ruminant Research vol 87 no 1ndash3 pp76ndash80 2009

[57] T Tylleskar M Banea N Bikangi G Nahimana L-A Perssonand H Rosling ldquoDietary determinants of a non-progressivespastic paraparesis (Konzo) a case-referent study in a highincidence area of Zairerdquo International Journal of Epidemiologyvol 24 no 5 pp 949ndash956 1995

[58] M M Swindle A Makin A J Herron F J Clubb Jr and K SFrazier ldquoSwine asmodels in biomedical research and toxicologytestingrdquo Veterinary Pathology vol 49 no 2 pp 344ndash356 2012

[59] B P Kamalu ldquoThe effect of a nutritionally-balanced cassava(Manihot esculenta Crantz) diet on endocrine function usingthe dog as a model 1 Pancreasrdquo British Journal of Nutrition vol65 no 3 pp 365ndash372 1991

[60] N P Okolie and A U Osagie ldquoDifferential effects of chroniccyanide intoxication on heart lung and pancreatic tissuesrdquo Foodand Chemical Toxicology vol 38 no 6 pp 543ndash548 2000

[61] M M Swindle and A C Smith ldquoComparative anatomy andphysiology of the pigrdquo Scandinavian Journal of LaboratoryAnimal Science vol 25 no 1 pp 11ndash21 1998

[62] L Jovanovic andD J Pettitt ldquoGestational diabetesmellitusrdquoTheJournal of the AmericanMedical Association vol 286 no 20 pp2516ndash2518 2001

[63] TA Buchanan andAHXiang ldquoGestational diabetesmellitusrdquoThe Journal of Clinical Investigation vol 115 no 3 pp 485ndash4912005

[64] The Practice Committee of American Society for ReproductiveMedicine ldquoSmoking and infertilityrdquo Fertility and Sterility vol4 pp 1181ndash1186 2004

[65] L Dencker R DrsquoArgy B R G Danielsson H Ghantous and GO Sperber ldquoSaturable accumulation of retinoic acid in neuraland neural crest derived cells in early embryonic developmentrdquoDevelopmental Pharmacology andTherapeutics vol 10 no 3 pp212ndash223 1987

[66] S Schwartz ldquoProviding toxicokinetic support for reproductivetoxicology studies in pharmaceutical developmentrdquo Archives ofToxicology vol 75 no 7 pp 381ndash387 2001

[67] W J Scott and H Nau ldquoAccumulation of weak acids in theyoung mammalian embryordquo in Pharmacokinetics in Teratoge-nesis H Nau and W J Scott Eds pp 72ndash76 CRC Press BocaRaton Fla USA 1987

[68] O O Tewe J H Maner and G Gomez ldquoInfluence of cassavadiets on placental thiocyanate transfer tissue rhodanese activityand performance of rats during gestationrdquo Journal of the Scienceof Food and Agriculture vol 28 no 8 pp 750ndash756 1977

[69] L Claudio C F Bearer andDWallinga ldquoAssessment of theUSenvironmental protection agency methods for identification ofhazards to developing organisms part II the developing toxicitytesting guidelinerdquo American Journal of Industrial Medicine vol35 no 6 pp 554ndash563 1999

4 Journal of Toxicology

Table 1 Reproductive parameters from sows treated with KCN from day 21 of pregnancy to term and the body weight of their piglets at birthuntil 120 days old

Parameters Control (119899 = 6) KCN (mgkg per day)20 (119899 = 6) 40 (119899 = 6) 60 (119899 = 6)

Body weight gain (kg) during pregnancya 696 plusmn 46 552 plusmn 51 498 plusmn 41 594 plusmn 82Number of assisted births 0 (5)b 0 (5) 4 (5) 1 (5)Live piglets 57 55 36 51

Male 27 30 13 24Female 30 25 23 27

Number of dead piglets 6 4 23lowast 10

Stillbirths 5 4 14 7Mummified fetuses 1 0 9 3

Subsequent total born piglets (mean) 126 118 72 94Piglets body weight at birtha 168 plusmn 004 (57)c 164 plusmn 003 (55) 171 plusmn 007 (36) 160 plusmn 005 (51)Overall weight gain (kg) of piglets at 120 days

Males 5525 plusmn 382 5247 plusmn 243 5407 plusmn 297 5279 plusmn 349Females 5382 plusmn 324 5235 plusmn 278 5162 plusmn 197 5162 plusmn 299

aMean plusmn SEMbNumber of deliveriescNumber of piglets weighedlowast

119875 lt 005 compared with controls

0

50

100

150

200

250

KCN0KCN2

KCN4KCN6

21 50 80 110Days of pregnancy

Thio

cyan

ate (

120583m

olL

)

(a)

KCN0KCN2

KCN4KCN6

0

10

20

30

40

50

Colostrum

Thio

cyan

ate (

120583m

olL

)

(b)

Figure 1 Thiocyanate levels (means plusmn SEM) in plasma (a) and colostrum (b) of sows allocated into four treatment groups KCN0 KCN2KCN4 andKCN6 that received respectively 00 20 40 and 60mgkg of bodyweight of cyanide fromday 21 of pregnancy to term 119901 lt 005compared to control

(data not shown) At PND120 there was no treatment effecton the overall weight gain of male or female piglets (Table 1)No histopathological lesions were observed in euthanizednewborns At PND120 piglets in all experimental groups hadsimilar histopathological changes as their mothers includingthyroid follicular enlargement and epithelial thickening andvacuolization (Figure 3)

4 Discussion

The administration of KCN orally was chosen aiming tomimic the main route of natural exposure to this speciesThedoses of KCN used here were chosen based on data from anearlier study conducted in our laboratory on male growing-finishing swine [31] inwhich the animals showed toxic effects

Journal of Toxicology 5

(b) (c)

(a)

Figure 2 Light photomicrograph of pregnant sows CNS (a) animal from control group ((b) and (c) resp) animals treated with 40 and60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing Purkinje cells with acidophilic cytoplasm (arrow (b)) andvacuolar degeneration of Purkinje cells (arrow (c)) Magnification times20 HE

(a) (b)

(c) (d)

Figure 3 Light photomicrograph of pregnant sows thyroid ((a) and (c)) animals from control group ((b) and (d)) animals treated with60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing enlargement of thyroid follicles (b) thickening of thefollicular epithelium (arrow (d)) and reabsorption vacuoles in colloid (arrowhead (d)) Magnification times4 ((a) and (b)) times20 ((c) and (d))HE

of chronic exposure to cyanide but did not manifest anyclinical signs of acute toxicity (eg dyspnea and convulsiveseizures) Similarly none of the pregnant females in thisstudy showed clinical symptoms of acute cyanide intoxicationduring the period of KCN administration

Given that the bitter taste of cyanide could compromisefood consumption during the study [45] starch biscuitsknown to be highly palatable to swine were chosen toborne the cyanide This procedure ensured that all femalesingested the target doses of cyanide quickly (usually in lessthan 2min) in each administration In a previous study oftoxicokinetics in pregnant sows we verified that following

oral exposure to KCN serum thiocyanate levels increase by3 hrs peak after 6 hrs and remain elevated for up to 21 hrs[46] Thus we assumed that the administration of cyanidetwice daily would ensure high serum levels of thiocyanatesteadily independently of the phase of pregnancy

Although some studies have found an associationbetween the reduction of body weight and prolonged expo-sure to cyanide in humans and animals [47ndash49] we did notdetect a decrease in this parameter in any of the pregnant sowsexposed to KCN This result corresponds with our previouswork conducted in growing-finishing pigs [31] Likewisestudies evaluating cyanide exposure during pregnancy in

6 Journal of Toxicology

(a)

(b) (c)

Figure 4 Light photomicrograph of pregnant sows kidney (a) animal from control group ((b) and (c) resp) animals treated with 20 and60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing vacuolar degeneration of the renal tubular epithelium(arrow (b) and (c)) Magnification times20 HE

goats [39] and rats [40] have not found differences in weightgain between the control and experimental animal groups Inthe same manner Tewe and Maner [50] did not find weightchanges in pregnant pigs fed cassava containing high levels ofcyanide

Although no biochemical changes were observed in preg-nant females exposed to KCN the histopathology performedin these sows revealed kidney and liver damage Lesionssimilar to those observed in our study have previously beendescribed in studies of other species chronically exposed tocyanide [30 51 52] as well as in pigs [31] It remains tobe determined if these lesions are a consequence of toxicitydirectly from cyanide thiocyanate or both

Studies clearly show that the CNS is an important targetin prolonged exposure to cyanide in both humans [53] andanimals [54ndash56] In humans it has been described to resultin several neuropathies such as spastic paraparesis (alsocalled ldquokonzordquo) Parkinsonrsquos disease cognitive impairmentand ocular pathologies [11 13 14 16 24 57]

The pig is an emerging model system for studying thehuman CNS [58] In a previous study in our lab where cya-nide was administered to growing pigs during the sameperiod evaluated here we found Purkinje cell degenerationand loss of cerebellar white matter in all groups treatedwith KCN [31] Similar lesions were found in this studyin sows exposed to cyanide These findings strengthen ourassertion that pigs should be used as an animal model tobetter understand the mechanism(s) of neurotoxicity fromcyanide andor its main metabolite

Tropical pancreatic diabetes has been associated withchronic cyanide exposure through consumption of cassavain man [25] and was reproduced in studies of dogs fedcassava [59] However studies administering cyanide todifferent animal species [29 60] including pigs [31] were

not able to reproduce these diabetogenic effects despite theanimals having islet cells functionality similar to humans[61] Given that pregnancy itself has a diabetogenic effectdue to a decrease in insulin sensitivity of maternal tissuesand resultant increase in insulin demand [62 63] pregnantsows would theoretically be evenmore susceptible to developdiabetes upon cyanide exposure Thus we hypothesized thatour study would have offered all the conditions needed toreproduce tropical pancreatic diabetes in pigs However wedid not find any changes in the parameters chosen to evaluatethe diabetogenic effects of cyanide (ie glucose levels andhistopathology) As we have previously shown that pregnantrats exposed to cyanide have increased levels of glucose andpancreatic islet degeneration [40] pregnant rats may be abetter animal model to assess the pancreotoxic effects ofcyanide

Studies have found a correlation between low doses ofchronic cyanide intake with a higher incidence of abortion inmares [32] sheep [36] and goats [39] Chronic cyanide intakein low doses from tobacco smoke has similarly been associ-ated with spontaneous abortion in women [64] although itremains unclear if this finding is specifically due to cyaniderather than other toxic compounds found in cigarettes

Higher rates of fetal death stillbirth assisted birth andmummified piglets were found in sows exposed to cyanidein this study The most common infectious causes of repro-ductive disease in Brazil (Campylobacter sp Mycobacteriumavium Brucella suis and Leptospira sp) were ruled out inthese animals Many causes could be posited to explain thesefinding but it is plausible to consider KCN as the mostlikely one We therefore suggest that the pig is a good animalmodel system for studying the role of cyanide andor its mainmetabolite in smoking-related abortions in women In ourstudy as we did not detect malformations in newborn pigs

Journal of Toxicology 7

it was not possible to conclude if ultrasound evaluation inpigs could be valuable for early detection of malformationsldquoin uterordquo

Amniocenteses in this study revealed high levels ofthiocyanate in the amniotic fluid of pregnant sows Denckeret al [65] and Schwartz [66] show that anionic substancessuch as the weak acid cyanuric acid can accumulate in theembryo and fetus This may be due to the pH gradient inmaternal plasma which is more acidic than the pH of fetalplasma Studies have thus observed an ldquoimprisonmentrdquo ofcyanide in the fetal organism [67] In addition it is knownthat the enzyme rhodanese is found in the placenta and thefetus [68] Thus it is possible that the ldquoarrestedrdquo cyanidecan be transformed into its main metabolite resulting inindirect thiocyanate toxicity to the fetus This hypothesisis additionally supported by a study in which cyanide orthiocyanate was administered to pregnant rats and bothsubstances were found to promote toxic effects in the fetuses[40]

The protocols that have been used to assess develop-mental toxicity in previous studies have certain limitations[69] One such limitation is the lack of postnatal monitoringof the development of the offspring which can result inerroneous conclusions about the developmental toxicity ofthe substances under examination In fact de Sousa et al [40]evaluated prenatal exposure to cyanide in rats and found thatalterations in pups were detected only at weaning Our studysupports these findings as we identified certain toxic effectsof cyanide in the piglets only at weaning

5 Conclusion

In conclusion pregnant sows exposed to cyanide had toxiceffects in both mothers and piglets However the toxiceffects in the offspring were observed belatedly Thereforethe data presented here reinforces the hypothesis that acomplementary evaluation of the neonate after weaning is anecessary component of studies on developmental toxicityIn addition we suggest that swine can serve as a usefulanimal model to study the neurological goitrogenic andreproductive toxicity of cyanide

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] W C Beamer R M Shealy and D S Prough ldquoAcute cyanidepoisoning from laetrile ingestionrdquo Annals of Emergency Medi-cine vol 12 no 7 pp 449ndash451 1983

[2] R Froldi M Cingolani and C Cacaci ldquoA case of suicide byingestion of sodium nitroprussiderdquo Journal of Forensic Sciencesvol 46 no 6 pp 1504ndash1506 2001

[3] J E Poulton ldquoCyanogenic compounds in plants and their toxiceffectsrdquo inHandbook of Natural Toxins Plant and Fungal ToxinsR F Keeler and A R Tu Eds vol 1 pp 117ndash157 Marcel DekkerNew York NY USA 1983

[4] United States Army Medical Research Institute of ChemicalDefense and Chemical Casualty Care Division ldquoCyaniderdquo inMedicalManagement of Chemical Causalities Handbook UnitedStates Army Medical Research Institute of Chemical DefenseAberdeen Md USA 3rd edition 2004

[5] M J Koschel ldquoWhere therersquos smoke there may be cyaniderdquoTheAmerican Journal of Nursing vol 102 no 8 pp 39ndash42 2002

[6] K Anseeuw N Delvau G Burillo-Putze et al ldquoCyanide poi-soning by fire smoke inhalation a European expert consensusrdquoEuropean Journal of Emergency Medicine vol 20 no 1 pp 2ndash92013

[7] V Schulz ldquoClinical pharmacokinetics- of nitroprusside cya-nide thiosulphate and thiocyanaterdquo Clinical Pharmacokineticsvol 9 no 3 pp 239ndash251 1984

[8] J Vetter ldquoPlant cyanogenic glycosidesrdquo Toxicon vol 38 no 1pp 11ndash36 2000

[9] B Ballantyne ldquoToxicology of cyanidesrdquo in Clinical and Exper-imental Toxicology of Cyanides B Ballantyne and T C MarrsEds pp 41ndash126 Wright Bristol UK 1987

[10] H Nzwalo and J Cliff ldquoKonzo from poverty cassava andcyanogen intake to toxico-nutritional neurological diseaserdquoPLoS Neglected Tropical Diseases vol 5 no 6 Article ID e10512011

[11] B O Osuntokun ldquoCassava diet chronic cyanide intoxicationand neuropathy in the Nigerian Africansrdquo World Review ofNutrition and Dietetics vol 36 pp 141ndash173 1981

[12] J Njoh ldquoTropical ataxic neuropathy in Liberiansrdquo Tropical andGeographical Medicine vol 42 no 1 pp 92ndash94 1990

[13] N L Rosenberg J A Myers and W R Wayne Martin ldquoCya-nide-induced parkinsonism clinical MRI and 6-fluorodopaPET studiesrdquo Neurology vol 39 no 1 pp 142ndash144 1989

[14] M Di Filippo N Tambasco G Muzi et al ldquoParkinsonism andcognitive impairment following chronic exposure to potassiumcyaniderdquoMovement Disorders vol 23 no 3 pp 468ndash470 2008

[15] A R Pettigrew and G S Fell ldquoSimplified colorimetric determi-nation of thiocyanate in biological fluids and its application toinvestigation of the toxic amblyopiasrdquo Clinical Chemistry vol18 no 9 pp 996ndash1000 1972

[16] Y Solberg M Rosner and M Belkin ldquoThe association betwencigarette smoking and ocular diseasesrdquo Survey of Ophthalmol-ogy vol 42 no 6 pp 535ndash547 1998

[17] A G Freeman ldquoOptic neuropathy and chronic cyanide intox-ication a reviewrdquo Journal of the Royal Society of Medicine vol81 no 2 pp 103ndash106 1988

[18] I G Syme J Bronte-Stewart and W S Foulds ldquoClinical andbiochemical findings in Leberrsquos hereditary optic atrophyrdquoTrans-actions of the Ophthalmological Societies of the United Kingdomvol 103 no 5 pp 556ndash559 1983

[19] K Tsao P A Aitken and D R Johns ldquoSmoking as an aetio-logical factor in a pedigree with Leberrsquos hereditary optic neu-ropathyrdquo British Journal of Ophthalmology vol 83 no 5 pp577ndash581 1999

[20] S J Crews ldquoBilateral amblyopia in west Indiansrdquo Transactionsof the Ophthalmological Societies of the United Kingdom vol 83pp 653ndash667 1963

[21] A D Mackenzie and C I Phillips ldquoWest Indian amblyopiardquoBrain vol 91 no 2 pp 249ndash260 1968

[22] F D Carroll ldquoJamaican optic neuropathy in immigrants to theUnited StatesrdquoThe American Journal of Ophthalmology vol 71no 1 pp 261ndash265 1971

8 Journal of Toxicology

[23] B O Osuntokun and O Osuntokun ldquoTropical amblyopia inNigeriansrdquo American Journal of Ophthalmology vol 72 no 4pp 708ndash716 1971

[24] K Tucker and T R Hedges ldquoFood shortages and an epidemicof optic and peripheral neuropathy in CubardquoNutrition Reviewsvol 51 no 12 pp 349ndash357 1993

[25] D E McMillan and P J Geevarghese ldquoDietary cyanide andtropical malnutrition diabetesrdquo Diabetes Care vol 2 no 2 pp202ndash208 1979

[26] C S Pitchumoni N K Jain A B Lowenfels and E P DimagnoldquoChronic cyanide poisoning unifying concept for alcoholic andtropical pancreatitisrdquo Pancreas vol 3 no 2 pp 220ndash222 1988

[27] B P Kamalu ldquoPathological changes in growing dogs fed ona balanced cassava (Manihot esculenta Crantz) dietrdquo BritishJournal of Nutrition vol 69 no 3 pp 921ndash934 1993

[28] B P Kamalu ldquoThe adverse effects of long-term cassava (Mani-hot esculenta Crantz) consumptionrdquo International Journal ofFood Sciences and Nutrition vol 46 no 1 pp 65ndash93 1995

[29] B Soto-Blanco S L Gorniak and E T Kimura ldquoPhysiopatho-logical effects of the administration of chronic cyanide togrowing goatsmdasha model for ingestion of cyanogenic plantsrdquoVeterinary ResearchCommunications vol 25 no 5 pp 379ndash3892001

[30] A B Sousa B Soto-Blanco J L Guerra E T Kimura and SL Gorniak ldquoDoes prolonged oral exposure to cyanide promotehepatotoxicity and nephrotoxicityrdquo Toxicology vol 174 no 2pp 87ndash95 2002

[31] H Manzano A B de Sousa B Soto-Blanco J L Guerra P CMaiorka and S L Gorniak ldquoEffects of long-term cyanide inges-tion by pigsrdquo Veterinary Research Communications vol 31 no1 pp 93ndash104 2007

[32] J T Prichard and J L Voss ldquoFetal ankylosis in horses associatedwith hybrid Sudan pasturerdquo Journal of the American VeterinaryMedical Association vol 150 no 8 pp 871ndash873 1967

[33] L A Selby R W Menges E C Houser R E Flatt and A ACase ldquoOutbreak of swine malformations associated with thewild black cherry Prunus serotinardquo Archives of EnvironmentalHealth vol 22 no 4 pp 496ndash501 1971

[34] M G W Rodel ldquoEffects of different grasses on the incidence ofneonatal goiter and skeletal deformities in autumn born lambsrdquoRhodesia Agricultural Journal vol 69 pp 59ndash60 1972

[35] J T Seaman M G Smeal and J C Wright ldquoThe possibleassociation of a sorghum (Sorghum sudanese) hybrid as acause of developmental defects in calvesrdquo Australian VeterinaryJournal vol 57 no 7 pp 351ndash352 1981

[36] G A Bradley H C Metcalf C Reggiardo et al ldquoNeuroaxonaldegeneration in sheep grazing Sorghum pasturesrdquo Journal ofVeterinary Diagnostic Investigation vol 7 no 2 pp 229ndash2361995

[37] J D Singh ldquoThe teratogenic effects of dietary cassava on thepregnant albino rat a preliminary reportrdquo Teratology vol 24no 3 pp 289ndash291 1981

[38] H K Kumbnani A Singh G Singh and P Parimoo ldquoLimbrsquosmalformations in SouthernNigeriamdashGarri (Manihot esculenta)a probable causerdquo Journal of Human Ecology vol 1 pp 189ndash1911990

[39] B Soto-Blanco and S L Gorniak ldquoPrenatal toxicity of cyanidein goatsmdasha model for teratological studies in ruminantsrdquoTheriogenology vol 62 no 6 pp 1012ndash1026 2004

[40] A B de Sousa P CMaiorka I DGoncalves L RM de Sa andS L Gorniak ldquoEvaluation of effects of prenatal exposure to the

cyanide and thiocyanate inwistar ratsrdquoReproductive Toxicologyvol 23 no 4 pp 568ndash577 2007

[41] N C Ganderup W Harvey J T Mortensen and W HarroukldquoTheminipig as nonrodent species in toxicologymdashwhere are wenowrdquo International Journal of Toxicology vol 31 no 6 pp 507ndash528 2012

[42] G Bode P Clausing F Gervais et al ldquoThe utility of theminipigas an animal model in regulatory toxicologyrdquo Journal of Phar-macological and Toxicological Methods vol 62 no 3 pp 196ndash220 2010

[43] R D Geisert R H Renegar W W Thatcher R M Robertsand FW Bazer ldquoEstablishment of pregnancy in the pig I Inter-relationships between preimplantation development of the pigblastocyst and uterine endometrial secretionsrdquoBiology of Repro-duction vol 27 no 4 pp 925ndash939 1982

[44] K T Szabo Congenital Malformations in Laboratory and FarmAnimals Academic Press San Diego Calif USA 1989

[45] E A Iyayi ldquoDietary cyanide effect on performance and serumtestosterone of growing male pigsrdquo Beitrage zur TropischenLandwirtschaft undVeterinarmedizin vol 29 no 3 pp 347ndash3521991

[46] H Manzano A B Souza and S L Gorniak ldquoExposicaocianıdrica em suınos uma abordagem dos parametros toxic-ocineticos utilizando o tiocianato como biomarcadorrdquoBrazilianJournal of Veterinary Research and Animal Science vol 43 pp93ndash101 2006

[47] P Blanc M Hogan K Mallin D Hryhorczuk S Hessl and BBernard ldquoCyanide intoxication among silver-reclaiming work-ersrdquo The Journal of the American Medical Association vol 253no 3 pp 367ndash371 1985

[48] C F I Onwuka ldquoHydrocyanic acid contents of tropical browseand their influence on performance of goatsrdquo Food Chemistryvol 45 no 1 pp 5ndash10 1992

[49] C Ibebunjo B P Kamalu and E C Ihemelandu ldquoComparisonof the effects of cassava (Manihot esculenta Crantz) organiccyanide and inorganic cyanide on muscle and bone develop-ment in a Nigerian breed of dogrdquo British Journal of Nutritionvol 68 no 2 pp 483ndash491 1992

[50] O O Tewe and J H Maner ldquoPerformance and pathophysio-logical changes in pregnant pigs fed cassava diets containingdifferent levels of cyaniderdquo Research in Veterinary Science vol30 no 2 pp 147ndash151 1981

[51] N P Okolie and A U Osagie ldquoLiver and kidney lesions andassociated enzyme changes induced in rabbits by chronic cya-nide exposurerdquo Food and Chemical Toxicology vol 37 no 7 pp745ndash750 1999

[52] B Soto-Blanco and S L Gorniak ldquoToxic effects of prolongedadministration of leaves of cassava (Manihot esculenta Crantz)to goatsrdquo Experimental and Toxicologic Pathology vol 62 no 4pp 361ndash366 2010

[53] J Wilson ldquoCyanide in human disease a review of clinical andlaboratory evidencerdquo Fundamental and Applied Toxicology vol3 no 5 pp 397ndash399 1983

[54] E M Mills P G Gunasekar L Li J L Borowitz and G EIsom ldquoDifferential susceptibility of brain areas cyanide involvesdifferent modes of cell deathrdquo Toxicology and Applied Pharma-cology vol 156 no 1 pp 6ndash16 1999

[55] B Soto-Blanco P CesarMarioka and S Lima Gorniak ldquoEffectsof long-term low-dose cyanide administration to ratsrdquo Ecotoxi-cology and Environmental Safety vol 53 no 1 pp 37ndash41 2002

Journal of Toxicology 9

[56] B Soto-Blanco F T V Pereira A F D CarvalhoM AMiglinoand S L Gorniak ldquoFetal andmaternal lesions of cyanide dosingto pregnant goatsrdquo Small Ruminant Research vol 87 no 1ndash3 pp76ndash80 2009

[57] T Tylleskar M Banea N Bikangi G Nahimana L-A Perssonand H Rosling ldquoDietary determinants of a non-progressivespastic paraparesis (Konzo) a case-referent study in a highincidence area of Zairerdquo International Journal of Epidemiologyvol 24 no 5 pp 949ndash956 1995

[58] M M Swindle A Makin A J Herron F J Clubb Jr and K SFrazier ldquoSwine asmodels in biomedical research and toxicologytestingrdquo Veterinary Pathology vol 49 no 2 pp 344ndash356 2012

[59] B P Kamalu ldquoThe effect of a nutritionally-balanced cassava(Manihot esculenta Crantz) diet on endocrine function usingthe dog as a model 1 Pancreasrdquo British Journal of Nutrition vol65 no 3 pp 365ndash372 1991

[60] N P Okolie and A U Osagie ldquoDifferential effects of chroniccyanide intoxication on heart lung and pancreatic tissuesrdquo Foodand Chemical Toxicology vol 38 no 6 pp 543ndash548 2000

[61] M M Swindle and A C Smith ldquoComparative anatomy andphysiology of the pigrdquo Scandinavian Journal of LaboratoryAnimal Science vol 25 no 1 pp 11ndash21 1998

[62] L Jovanovic andD J Pettitt ldquoGestational diabetesmellitusrdquoTheJournal of the AmericanMedical Association vol 286 no 20 pp2516ndash2518 2001

[63] TA Buchanan andAHXiang ldquoGestational diabetesmellitusrdquoThe Journal of Clinical Investigation vol 115 no 3 pp 485ndash4912005

[64] The Practice Committee of American Society for ReproductiveMedicine ldquoSmoking and infertilityrdquo Fertility and Sterility vol4 pp 1181ndash1186 2004

[65] L Dencker R DrsquoArgy B R G Danielsson H Ghantous and GO Sperber ldquoSaturable accumulation of retinoic acid in neuraland neural crest derived cells in early embryonic developmentrdquoDevelopmental Pharmacology andTherapeutics vol 10 no 3 pp212ndash223 1987

[66] S Schwartz ldquoProviding toxicokinetic support for reproductivetoxicology studies in pharmaceutical developmentrdquo Archives ofToxicology vol 75 no 7 pp 381ndash387 2001

[67] W J Scott and H Nau ldquoAccumulation of weak acids in theyoung mammalian embryordquo in Pharmacokinetics in Teratoge-nesis H Nau and W J Scott Eds pp 72ndash76 CRC Press BocaRaton Fla USA 1987

[68] O O Tewe J H Maner and G Gomez ldquoInfluence of cassavadiets on placental thiocyanate transfer tissue rhodanese activityand performance of rats during gestationrdquo Journal of the Scienceof Food and Agriculture vol 28 no 8 pp 750ndash756 1977

[69] L Claudio C F Bearer andDWallinga ldquoAssessment of theUSenvironmental protection agency methods for identification ofhazards to developing organisms part II the developing toxicitytesting guidelinerdquo American Journal of Industrial Medicine vol35 no 6 pp 554ndash563 1999

Journal of Toxicology 5

(b) (c)

(a)

Figure 2 Light photomicrograph of pregnant sows CNS (a) animal from control group ((b) and (c) resp) animals treated with 40 and60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing Purkinje cells with acidophilic cytoplasm (arrow (b)) andvacuolar degeneration of Purkinje cells (arrow (c)) Magnification times20 HE

(a) (b)

(c) (d)

Figure 3 Light photomicrograph of pregnant sows thyroid ((a) and (c)) animals from control group ((b) and (d)) animals treated with60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing enlargement of thyroid follicles (b) thickening of thefollicular epithelium (arrow (d)) and reabsorption vacuoles in colloid (arrowhead (d)) Magnification times4 ((a) and (b)) times20 ((c) and (d))HE

of chronic exposure to cyanide but did not manifest anyclinical signs of acute toxicity (eg dyspnea and convulsiveseizures) Similarly none of the pregnant females in thisstudy showed clinical symptoms of acute cyanide intoxicationduring the period of KCN administration

Given that the bitter taste of cyanide could compromisefood consumption during the study [45] starch biscuitsknown to be highly palatable to swine were chosen toborne the cyanide This procedure ensured that all femalesingested the target doses of cyanide quickly (usually in lessthan 2min) in each administration In a previous study oftoxicokinetics in pregnant sows we verified that following

oral exposure to KCN serum thiocyanate levels increase by3 hrs peak after 6 hrs and remain elevated for up to 21 hrs[46] Thus we assumed that the administration of cyanidetwice daily would ensure high serum levels of thiocyanatesteadily independently of the phase of pregnancy

Although some studies have found an associationbetween the reduction of body weight and prolonged expo-sure to cyanide in humans and animals [47ndash49] we did notdetect a decrease in this parameter in any of the pregnant sowsexposed to KCN This result corresponds with our previouswork conducted in growing-finishing pigs [31] Likewisestudies evaluating cyanide exposure during pregnancy in

6 Journal of Toxicology

(a)

(b) (c)

Figure 4 Light photomicrograph of pregnant sows kidney (a) animal from control group ((b) and (c) resp) animals treated with 20 and60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing vacuolar degeneration of the renal tubular epithelium(arrow (b) and (c)) Magnification times20 HE

goats [39] and rats [40] have not found differences in weightgain between the control and experimental animal groups Inthe same manner Tewe and Maner [50] did not find weightchanges in pregnant pigs fed cassava containing high levels ofcyanide

Although no biochemical changes were observed in preg-nant females exposed to KCN the histopathology performedin these sows revealed kidney and liver damage Lesionssimilar to those observed in our study have previously beendescribed in studies of other species chronically exposed tocyanide [30 51 52] as well as in pigs [31] It remains tobe determined if these lesions are a consequence of toxicitydirectly from cyanide thiocyanate or both

Studies clearly show that the CNS is an important targetin prolonged exposure to cyanide in both humans [53] andanimals [54ndash56] In humans it has been described to resultin several neuropathies such as spastic paraparesis (alsocalled ldquokonzordquo) Parkinsonrsquos disease cognitive impairmentand ocular pathologies [11 13 14 16 24 57]

The pig is an emerging model system for studying thehuman CNS [58] In a previous study in our lab where cya-nide was administered to growing pigs during the sameperiod evaluated here we found Purkinje cell degenerationand loss of cerebellar white matter in all groups treatedwith KCN [31] Similar lesions were found in this studyin sows exposed to cyanide These findings strengthen ourassertion that pigs should be used as an animal model tobetter understand the mechanism(s) of neurotoxicity fromcyanide andor its main metabolite

Tropical pancreatic diabetes has been associated withchronic cyanide exposure through consumption of cassavain man [25] and was reproduced in studies of dogs fedcassava [59] However studies administering cyanide todifferent animal species [29 60] including pigs [31] were

not able to reproduce these diabetogenic effects despite theanimals having islet cells functionality similar to humans[61] Given that pregnancy itself has a diabetogenic effectdue to a decrease in insulin sensitivity of maternal tissuesand resultant increase in insulin demand [62 63] pregnantsows would theoretically be evenmore susceptible to developdiabetes upon cyanide exposure Thus we hypothesized thatour study would have offered all the conditions needed toreproduce tropical pancreatic diabetes in pigs However wedid not find any changes in the parameters chosen to evaluatethe diabetogenic effects of cyanide (ie glucose levels andhistopathology) As we have previously shown that pregnantrats exposed to cyanide have increased levels of glucose andpancreatic islet degeneration [40] pregnant rats may be abetter animal model to assess the pancreotoxic effects ofcyanide

Studies have found a correlation between low doses ofchronic cyanide intake with a higher incidence of abortion inmares [32] sheep [36] and goats [39] Chronic cyanide intakein low doses from tobacco smoke has similarly been associ-ated with spontaneous abortion in women [64] although itremains unclear if this finding is specifically due to cyaniderather than other toxic compounds found in cigarettes

Higher rates of fetal death stillbirth assisted birth andmummified piglets were found in sows exposed to cyanidein this study The most common infectious causes of repro-ductive disease in Brazil (Campylobacter sp Mycobacteriumavium Brucella suis and Leptospira sp) were ruled out inthese animals Many causes could be posited to explain thesefinding but it is plausible to consider KCN as the mostlikely one We therefore suggest that the pig is a good animalmodel system for studying the role of cyanide andor its mainmetabolite in smoking-related abortions in women In ourstudy as we did not detect malformations in newborn pigs

Journal of Toxicology 7

it was not possible to conclude if ultrasound evaluation inpigs could be valuable for early detection of malformationsldquoin uterordquo

Amniocenteses in this study revealed high levels ofthiocyanate in the amniotic fluid of pregnant sows Denckeret al [65] and Schwartz [66] show that anionic substancessuch as the weak acid cyanuric acid can accumulate in theembryo and fetus This may be due to the pH gradient inmaternal plasma which is more acidic than the pH of fetalplasma Studies have thus observed an ldquoimprisonmentrdquo ofcyanide in the fetal organism [67] In addition it is knownthat the enzyme rhodanese is found in the placenta and thefetus [68] Thus it is possible that the ldquoarrestedrdquo cyanidecan be transformed into its main metabolite resulting inindirect thiocyanate toxicity to the fetus This hypothesisis additionally supported by a study in which cyanide orthiocyanate was administered to pregnant rats and bothsubstances were found to promote toxic effects in the fetuses[40]

The protocols that have been used to assess develop-mental toxicity in previous studies have certain limitations[69] One such limitation is the lack of postnatal monitoringof the development of the offspring which can result inerroneous conclusions about the developmental toxicity ofthe substances under examination In fact de Sousa et al [40]evaluated prenatal exposure to cyanide in rats and found thatalterations in pups were detected only at weaning Our studysupports these findings as we identified certain toxic effectsof cyanide in the piglets only at weaning

5 Conclusion

In conclusion pregnant sows exposed to cyanide had toxiceffects in both mothers and piglets However the toxiceffects in the offspring were observed belatedly Thereforethe data presented here reinforces the hypothesis that acomplementary evaluation of the neonate after weaning is anecessary component of studies on developmental toxicityIn addition we suggest that swine can serve as a usefulanimal model to study the neurological goitrogenic andreproductive toxicity of cyanide

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] W C Beamer R M Shealy and D S Prough ldquoAcute cyanidepoisoning from laetrile ingestionrdquo Annals of Emergency Medi-cine vol 12 no 7 pp 449ndash451 1983

[2] R Froldi M Cingolani and C Cacaci ldquoA case of suicide byingestion of sodium nitroprussiderdquo Journal of Forensic Sciencesvol 46 no 6 pp 1504ndash1506 2001

[3] J E Poulton ldquoCyanogenic compounds in plants and their toxiceffectsrdquo inHandbook of Natural Toxins Plant and Fungal ToxinsR F Keeler and A R Tu Eds vol 1 pp 117ndash157 Marcel DekkerNew York NY USA 1983

[4] United States Army Medical Research Institute of ChemicalDefense and Chemical Casualty Care Division ldquoCyaniderdquo inMedicalManagement of Chemical Causalities Handbook UnitedStates Army Medical Research Institute of Chemical DefenseAberdeen Md USA 3rd edition 2004

[5] M J Koschel ldquoWhere therersquos smoke there may be cyaniderdquoTheAmerican Journal of Nursing vol 102 no 8 pp 39ndash42 2002

[6] K Anseeuw N Delvau G Burillo-Putze et al ldquoCyanide poi-soning by fire smoke inhalation a European expert consensusrdquoEuropean Journal of Emergency Medicine vol 20 no 1 pp 2ndash92013

[7] V Schulz ldquoClinical pharmacokinetics- of nitroprusside cya-nide thiosulphate and thiocyanaterdquo Clinical Pharmacokineticsvol 9 no 3 pp 239ndash251 1984

[8] J Vetter ldquoPlant cyanogenic glycosidesrdquo Toxicon vol 38 no 1pp 11ndash36 2000

[9] B Ballantyne ldquoToxicology of cyanidesrdquo in Clinical and Exper-imental Toxicology of Cyanides B Ballantyne and T C MarrsEds pp 41ndash126 Wright Bristol UK 1987

[10] H Nzwalo and J Cliff ldquoKonzo from poverty cassava andcyanogen intake to toxico-nutritional neurological diseaserdquoPLoS Neglected Tropical Diseases vol 5 no 6 Article ID e10512011

[11] B O Osuntokun ldquoCassava diet chronic cyanide intoxicationand neuropathy in the Nigerian Africansrdquo World Review ofNutrition and Dietetics vol 36 pp 141ndash173 1981

[12] J Njoh ldquoTropical ataxic neuropathy in Liberiansrdquo Tropical andGeographical Medicine vol 42 no 1 pp 92ndash94 1990

[13] N L Rosenberg J A Myers and W R Wayne Martin ldquoCya-nide-induced parkinsonism clinical MRI and 6-fluorodopaPET studiesrdquo Neurology vol 39 no 1 pp 142ndash144 1989

[14] M Di Filippo N Tambasco G Muzi et al ldquoParkinsonism andcognitive impairment following chronic exposure to potassiumcyaniderdquoMovement Disorders vol 23 no 3 pp 468ndash470 2008

[15] A R Pettigrew and G S Fell ldquoSimplified colorimetric determi-nation of thiocyanate in biological fluids and its application toinvestigation of the toxic amblyopiasrdquo Clinical Chemistry vol18 no 9 pp 996ndash1000 1972

[16] Y Solberg M Rosner and M Belkin ldquoThe association betwencigarette smoking and ocular diseasesrdquo Survey of Ophthalmol-ogy vol 42 no 6 pp 535ndash547 1998

[17] A G Freeman ldquoOptic neuropathy and chronic cyanide intox-ication a reviewrdquo Journal of the Royal Society of Medicine vol81 no 2 pp 103ndash106 1988

[18] I G Syme J Bronte-Stewart and W S Foulds ldquoClinical andbiochemical findings in Leberrsquos hereditary optic atrophyrdquoTrans-actions of the Ophthalmological Societies of the United Kingdomvol 103 no 5 pp 556ndash559 1983

[19] K Tsao P A Aitken and D R Johns ldquoSmoking as an aetio-logical factor in a pedigree with Leberrsquos hereditary optic neu-ropathyrdquo British Journal of Ophthalmology vol 83 no 5 pp577ndash581 1999

[20] S J Crews ldquoBilateral amblyopia in west Indiansrdquo Transactionsof the Ophthalmological Societies of the United Kingdom vol 83pp 653ndash667 1963

[21] A D Mackenzie and C I Phillips ldquoWest Indian amblyopiardquoBrain vol 91 no 2 pp 249ndash260 1968

[22] F D Carroll ldquoJamaican optic neuropathy in immigrants to theUnited StatesrdquoThe American Journal of Ophthalmology vol 71no 1 pp 261ndash265 1971

8 Journal of Toxicology

[23] B O Osuntokun and O Osuntokun ldquoTropical amblyopia inNigeriansrdquo American Journal of Ophthalmology vol 72 no 4pp 708ndash716 1971

[24] K Tucker and T R Hedges ldquoFood shortages and an epidemicof optic and peripheral neuropathy in CubardquoNutrition Reviewsvol 51 no 12 pp 349ndash357 1993

[25] D E McMillan and P J Geevarghese ldquoDietary cyanide andtropical malnutrition diabetesrdquo Diabetes Care vol 2 no 2 pp202ndash208 1979

[26] C S Pitchumoni N K Jain A B Lowenfels and E P DimagnoldquoChronic cyanide poisoning unifying concept for alcoholic andtropical pancreatitisrdquo Pancreas vol 3 no 2 pp 220ndash222 1988

[27] B P Kamalu ldquoPathological changes in growing dogs fed ona balanced cassava (Manihot esculenta Crantz) dietrdquo BritishJournal of Nutrition vol 69 no 3 pp 921ndash934 1993

[28] B P Kamalu ldquoThe adverse effects of long-term cassava (Mani-hot esculenta Crantz) consumptionrdquo International Journal ofFood Sciences and Nutrition vol 46 no 1 pp 65ndash93 1995

[29] B Soto-Blanco S L Gorniak and E T Kimura ldquoPhysiopatho-logical effects of the administration of chronic cyanide togrowing goatsmdasha model for ingestion of cyanogenic plantsrdquoVeterinary ResearchCommunications vol 25 no 5 pp 379ndash3892001

[30] A B Sousa B Soto-Blanco J L Guerra E T Kimura and SL Gorniak ldquoDoes prolonged oral exposure to cyanide promotehepatotoxicity and nephrotoxicityrdquo Toxicology vol 174 no 2pp 87ndash95 2002

[31] H Manzano A B de Sousa B Soto-Blanco J L Guerra P CMaiorka and S L Gorniak ldquoEffects of long-term cyanide inges-tion by pigsrdquo Veterinary Research Communications vol 31 no1 pp 93ndash104 2007

[32] J T Prichard and J L Voss ldquoFetal ankylosis in horses associatedwith hybrid Sudan pasturerdquo Journal of the American VeterinaryMedical Association vol 150 no 8 pp 871ndash873 1967

[33] L A Selby R W Menges E C Houser R E Flatt and A ACase ldquoOutbreak of swine malformations associated with thewild black cherry Prunus serotinardquo Archives of EnvironmentalHealth vol 22 no 4 pp 496ndash501 1971

[34] M G W Rodel ldquoEffects of different grasses on the incidence ofneonatal goiter and skeletal deformities in autumn born lambsrdquoRhodesia Agricultural Journal vol 69 pp 59ndash60 1972

[35] J T Seaman M G Smeal and J C Wright ldquoThe possibleassociation of a sorghum (Sorghum sudanese) hybrid as acause of developmental defects in calvesrdquo Australian VeterinaryJournal vol 57 no 7 pp 351ndash352 1981

[36] G A Bradley H C Metcalf C Reggiardo et al ldquoNeuroaxonaldegeneration in sheep grazing Sorghum pasturesrdquo Journal ofVeterinary Diagnostic Investigation vol 7 no 2 pp 229ndash2361995

[37] J D Singh ldquoThe teratogenic effects of dietary cassava on thepregnant albino rat a preliminary reportrdquo Teratology vol 24no 3 pp 289ndash291 1981

[38] H K Kumbnani A Singh G Singh and P Parimoo ldquoLimbrsquosmalformations in SouthernNigeriamdashGarri (Manihot esculenta)a probable causerdquo Journal of Human Ecology vol 1 pp 189ndash1911990

[39] B Soto-Blanco and S L Gorniak ldquoPrenatal toxicity of cyanidein goatsmdasha model for teratological studies in ruminantsrdquoTheriogenology vol 62 no 6 pp 1012ndash1026 2004

[40] A B de Sousa P CMaiorka I DGoncalves L RM de Sa andS L Gorniak ldquoEvaluation of effects of prenatal exposure to the

cyanide and thiocyanate inwistar ratsrdquoReproductive Toxicologyvol 23 no 4 pp 568ndash577 2007

[41] N C Ganderup W Harvey J T Mortensen and W HarroukldquoTheminipig as nonrodent species in toxicologymdashwhere are wenowrdquo International Journal of Toxicology vol 31 no 6 pp 507ndash528 2012

[42] G Bode P Clausing F Gervais et al ldquoThe utility of theminipigas an animal model in regulatory toxicologyrdquo Journal of Phar-macological and Toxicological Methods vol 62 no 3 pp 196ndash220 2010

[43] R D Geisert R H Renegar W W Thatcher R M Robertsand FW Bazer ldquoEstablishment of pregnancy in the pig I Inter-relationships between preimplantation development of the pigblastocyst and uterine endometrial secretionsrdquoBiology of Repro-duction vol 27 no 4 pp 925ndash939 1982

[44] K T Szabo Congenital Malformations in Laboratory and FarmAnimals Academic Press San Diego Calif USA 1989

[45] E A Iyayi ldquoDietary cyanide effect on performance and serumtestosterone of growing male pigsrdquo Beitrage zur TropischenLandwirtschaft undVeterinarmedizin vol 29 no 3 pp 347ndash3521991

[46] H Manzano A B Souza and S L Gorniak ldquoExposicaocianıdrica em suınos uma abordagem dos parametros toxic-ocineticos utilizando o tiocianato como biomarcadorrdquoBrazilianJournal of Veterinary Research and Animal Science vol 43 pp93ndash101 2006

[47] P Blanc M Hogan K Mallin D Hryhorczuk S Hessl and BBernard ldquoCyanide intoxication among silver-reclaiming work-ersrdquo The Journal of the American Medical Association vol 253no 3 pp 367ndash371 1985

[48] C F I Onwuka ldquoHydrocyanic acid contents of tropical browseand their influence on performance of goatsrdquo Food Chemistryvol 45 no 1 pp 5ndash10 1992

[49] C Ibebunjo B P Kamalu and E C Ihemelandu ldquoComparisonof the effects of cassava (Manihot esculenta Crantz) organiccyanide and inorganic cyanide on muscle and bone develop-ment in a Nigerian breed of dogrdquo British Journal of Nutritionvol 68 no 2 pp 483ndash491 1992

[50] O O Tewe and J H Maner ldquoPerformance and pathophysio-logical changes in pregnant pigs fed cassava diets containingdifferent levels of cyaniderdquo Research in Veterinary Science vol30 no 2 pp 147ndash151 1981

[51] N P Okolie and A U Osagie ldquoLiver and kidney lesions andassociated enzyme changes induced in rabbits by chronic cya-nide exposurerdquo Food and Chemical Toxicology vol 37 no 7 pp745ndash750 1999

[52] B Soto-Blanco and S L Gorniak ldquoToxic effects of prolongedadministration of leaves of cassava (Manihot esculenta Crantz)to goatsrdquo Experimental and Toxicologic Pathology vol 62 no 4pp 361ndash366 2010

[53] J Wilson ldquoCyanide in human disease a review of clinical andlaboratory evidencerdquo Fundamental and Applied Toxicology vol3 no 5 pp 397ndash399 1983

[54] E M Mills P G Gunasekar L Li J L Borowitz and G EIsom ldquoDifferential susceptibility of brain areas cyanide involvesdifferent modes of cell deathrdquo Toxicology and Applied Pharma-cology vol 156 no 1 pp 6ndash16 1999

[55] B Soto-Blanco P CesarMarioka and S Lima Gorniak ldquoEffectsof long-term low-dose cyanide administration to ratsrdquo Ecotoxi-cology and Environmental Safety vol 53 no 1 pp 37ndash41 2002

Journal of Toxicology 9

[56] B Soto-Blanco F T V Pereira A F D CarvalhoM AMiglinoand S L Gorniak ldquoFetal andmaternal lesions of cyanide dosingto pregnant goatsrdquo Small Ruminant Research vol 87 no 1ndash3 pp76ndash80 2009

[57] T Tylleskar M Banea N Bikangi G Nahimana L-A Perssonand H Rosling ldquoDietary determinants of a non-progressivespastic paraparesis (Konzo) a case-referent study in a highincidence area of Zairerdquo International Journal of Epidemiologyvol 24 no 5 pp 949ndash956 1995

[58] M M Swindle A Makin A J Herron F J Clubb Jr and K SFrazier ldquoSwine asmodels in biomedical research and toxicologytestingrdquo Veterinary Pathology vol 49 no 2 pp 344ndash356 2012

[59] B P Kamalu ldquoThe effect of a nutritionally-balanced cassava(Manihot esculenta Crantz) diet on endocrine function usingthe dog as a model 1 Pancreasrdquo British Journal of Nutrition vol65 no 3 pp 365ndash372 1991

[60] N P Okolie and A U Osagie ldquoDifferential effects of chroniccyanide intoxication on heart lung and pancreatic tissuesrdquo Foodand Chemical Toxicology vol 38 no 6 pp 543ndash548 2000

[61] M M Swindle and A C Smith ldquoComparative anatomy andphysiology of the pigrdquo Scandinavian Journal of LaboratoryAnimal Science vol 25 no 1 pp 11ndash21 1998

[62] L Jovanovic andD J Pettitt ldquoGestational diabetesmellitusrdquoTheJournal of the AmericanMedical Association vol 286 no 20 pp2516ndash2518 2001

[63] TA Buchanan andAHXiang ldquoGestational diabetesmellitusrdquoThe Journal of Clinical Investigation vol 115 no 3 pp 485ndash4912005

[64] The Practice Committee of American Society for ReproductiveMedicine ldquoSmoking and infertilityrdquo Fertility and Sterility vol4 pp 1181ndash1186 2004

[65] L Dencker R DrsquoArgy B R G Danielsson H Ghantous and GO Sperber ldquoSaturable accumulation of retinoic acid in neuraland neural crest derived cells in early embryonic developmentrdquoDevelopmental Pharmacology andTherapeutics vol 10 no 3 pp212ndash223 1987

[66] S Schwartz ldquoProviding toxicokinetic support for reproductivetoxicology studies in pharmaceutical developmentrdquo Archives ofToxicology vol 75 no 7 pp 381ndash387 2001

[67] W J Scott and H Nau ldquoAccumulation of weak acids in theyoung mammalian embryordquo in Pharmacokinetics in Teratoge-nesis H Nau and W J Scott Eds pp 72ndash76 CRC Press BocaRaton Fla USA 1987

[68] O O Tewe J H Maner and G Gomez ldquoInfluence of cassavadiets on placental thiocyanate transfer tissue rhodanese activityand performance of rats during gestationrdquo Journal of the Scienceof Food and Agriculture vol 28 no 8 pp 750ndash756 1977

[69] L Claudio C F Bearer andDWallinga ldquoAssessment of theUSenvironmental protection agency methods for identification ofhazards to developing organisms part II the developing toxicitytesting guidelinerdquo American Journal of Industrial Medicine vol35 no 6 pp 554ndash563 1999

6 Journal of Toxicology

(a)

(b) (c)

Figure 4 Light photomicrograph of pregnant sows kidney (a) animal from control group ((b) and (c) resp) animals treated with 20 and60mgkg of body weight of cyanide from day 21 of pregnancy to 110 term showing vacuolar degeneration of the renal tubular epithelium(arrow (b) and (c)) Magnification times20 HE

goats [39] and rats [40] have not found differences in weightgain between the control and experimental animal groups Inthe same manner Tewe and Maner [50] did not find weightchanges in pregnant pigs fed cassava containing high levels ofcyanide

Although no biochemical changes were observed in preg-nant females exposed to KCN the histopathology performedin these sows revealed kidney and liver damage Lesionssimilar to those observed in our study have previously beendescribed in studies of other species chronically exposed tocyanide [30 51 52] as well as in pigs [31] It remains tobe determined if these lesions are a consequence of toxicitydirectly from cyanide thiocyanate or both

Studies clearly show that the CNS is an important targetin prolonged exposure to cyanide in both humans [53] andanimals [54ndash56] In humans it has been described to resultin several neuropathies such as spastic paraparesis (alsocalled ldquokonzordquo) Parkinsonrsquos disease cognitive impairmentand ocular pathologies [11 13 14 16 24 57]

The pig is an emerging model system for studying thehuman CNS [58] In a previous study in our lab where cya-nide was administered to growing pigs during the sameperiod evaluated here we found Purkinje cell degenerationand loss of cerebellar white matter in all groups treatedwith KCN [31] Similar lesions were found in this studyin sows exposed to cyanide These findings strengthen ourassertion that pigs should be used as an animal model tobetter understand the mechanism(s) of neurotoxicity fromcyanide andor its main metabolite

Tropical pancreatic diabetes has been associated withchronic cyanide exposure through consumption of cassavain man [25] and was reproduced in studies of dogs fedcassava [59] However studies administering cyanide todifferent animal species [29 60] including pigs [31] were

not able to reproduce these diabetogenic effects despite theanimals having islet cells functionality similar to humans[61] Given that pregnancy itself has a diabetogenic effectdue to a decrease in insulin sensitivity of maternal tissuesand resultant increase in insulin demand [62 63] pregnantsows would theoretically be evenmore susceptible to developdiabetes upon cyanide exposure Thus we hypothesized thatour study would have offered all the conditions needed toreproduce tropical pancreatic diabetes in pigs However wedid not find any changes in the parameters chosen to evaluatethe diabetogenic effects of cyanide (ie glucose levels andhistopathology) As we have previously shown that pregnantrats exposed to cyanide have increased levels of glucose andpancreatic islet degeneration [40] pregnant rats may be abetter animal model to assess the pancreotoxic effects ofcyanide

Studies have found a correlation between low doses ofchronic cyanide intake with a higher incidence of abortion inmares [32] sheep [36] and goats [39] Chronic cyanide intakein low doses from tobacco smoke has similarly been associ-ated with spontaneous abortion in women [64] although itremains unclear if this finding is specifically due to cyaniderather than other toxic compounds found in cigarettes

Higher rates of fetal death stillbirth assisted birth andmummified piglets were found in sows exposed to cyanidein this study The most common infectious causes of repro-ductive disease in Brazil (Campylobacter sp Mycobacteriumavium Brucella suis and Leptospira sp) were ruled out inthese animals Many causes could be posited to explain thesefinding but it is plausible to consider KCN as the mostlikely one We therefore suggest that the pig is a good animalmodel system for studying the role of cyanide andor its mainmetabolite in smoking-related abortions in women In ourstudy as we did not detect malformations in newborn pigs

Journal of Toxicology 7

it was not possible to conclude if ultrasound evaluation inpigs could be valuable for early detection of malformationsldquoin uterordquo

Amniocenteses in this study revealed high levels ofthiocyanate in the amniotic fluid of pregnant sows Denckeret al [65] and Schwartz [66] show that anionic substancessuch as the weak acid cyanuric acid can accumulate in theembryo and fetus This may be due to the pH gradient inmaternal plasma which is more acidic than the pH of fetalplasma Studies have thus observed an ldquoimprisonmentrdquo ofcyanide in the fetal organism [67] In addition it is knownthat the enzyme rhodanese is found in the placenta and thefetus [68] Thus it is possible that the ldquoarrestedrdquo cyanidecan be transformed into its main metabolite resulting inindirect thiocyanate toxicity to the fetus This hypothesisis additionally supported by a study in which cyanide orthiocyanate was administered to pregnant rats and bothsubstances were found to promote toxic effects in the fetuses[40]

The protocols that have been used to assess develop-mental toxicity in previous studies have certain limitations[69] One such limitation is the lack of postnatal monitoringof the development of the offspring which can result inerroneous conclusions about the developmental toxicity ofthe substances under examination In fact de Sousa et al [40]evaluated prenatal exposure to cyanide in rats and found thatalterations in pups were detected only at weaning Our studysupports these findings as we identified certain toxic effectsof cyanide in the piglets only at weaning

5 Conclusion

In conclusion pregnant sows exposed to cyanide had toxiceffects in both mothers and piglets However the toxiceffects in the offspring were observed belatedly Thereforethe data presented here reinforces the hypothesis that acomplementary evaluation of the neonate after weaning is anecessary component of studies on developmental toxicityIn addition we suggest that swine can serve as a usefulanimal model to study the neurological goitrogenic andreproductive toxicity of cyanide

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] W C Beamer R M Shealy and D S Prough ldquoAcute cyanidepoisoning from laetrile ingestionrdquo Annals of Emergency Medi-cine vol 12 no 7 pp 449ndash451 1983

[2] R Froldi M Cingolani and C Cacaci ldquoA case of suicide byingestion of sodium nitroprussiderdquo Journal of Forensic Sciencesvol 46 no 6 pp 1504ndash1506 2001

[3] J E Poulton ldquoCyanogenic compounds in plants and their toxiceffectsrdquo inHandbook of Natural Toxins Plant and Fungal ToxinsR F Keeler and A R Tu Eds vol 1 pp 117ndash157 Marcel DekkerNew York NY USA 1983

[4] United States Army Medical Research Institute of ChemicalDefense and Chemical Casualty Care Division ldquoCyaniderdquo inMedicalManagement of Chemical Causalities Handbook UnitedStates Army Medical Research Institute of Chemical DefenseAberdeen Md USA 3rd edition 2004

[5] M J Koschel ldquoWhere therersquos smoke there may be cyaniderdquoTheAmerican Journal of Nursing vol 102 no 8 pp 39ndash42 2002

[6] K Anseeuw N Delvau G Burillo-Putze et al ldquoCyanide poi-soning by fire smoke inhalation a European expert consensusrdquoEuropean Journal of Emergency Medicine vol 20 no 1 pp 2ndash92013

[7] V Schulz ldquoClinical pharmacokinetics- of nitroprusside cya-nide thiosulphate and thiocyanaterdquo Clinical Pharmacokineticsvol 9 no 3 pp 239ndash251 1984

[8] J Vetter ldquoPlant cyanogenic glycosidesrdquo Toxicon vol 38 no 1pp 11ndash36 2000

[9] B Ballantyne ldquoToxicology of cyanidesrdquo in Clinical and Exper-imental Toxicology of Cyanides B Ballantyne and T C MarrsEds pp 41ndash126 Wright Bristol UK 1987

[10] H Nzwalo and J Cliff ldquoKonzo from poverty cassava andcyanogen intake to toxico-nutritional neurological diseaserdquoPLoS Neglected Tropical Diseases vol 5 no 6 Article ID e10512011

[11] B O Osuntokun ldquoCassava diet chronic cyanide intoxicationand neuropathy in the Nigerian Africansrdquo World Review ofNutrition and Dietetics vol 36 pp 141ndash173 1981

[12] J Njoh ldquoTropical ataxic neuropathy in Liberiansrdquo Tropical andGeographical Medicine vol 42 no 1 pp 92ndash94 1990

[13] N L Rosenberg J A Myers and W R Wayne Martin ldquoCya-nide-induced parkinsonism clinical MRI and 6-fluorodopaPET studiesrdquo Neurology vol 39 no 1 pp 142ndash144 1989

[14] M Di Filippo N Tambasco G Muzi et al ldquoParkinsonism andcognitive impairment following chronic exposure to potassiumcyaniderdquoMovement Disorders vol 23 no 3 pp 468ndash470 2008

[15] A R Pettigrew and G S Fell ldquoSimplified colorimetric determi-nation of thiocyanate in biological fluids and its application toinvestigation of the toxic amblyopiasrdquo Clinical Chemistry vol18 no 9 pp 996ndash1000 1972

[16] Y Solberg M Rosner and M Belkin ldquoThe association betwencigarette smoking and ocular diseasesrdquo Survey of Ophthalmol-ogy vol 42 no 6 pp 535ndash547 1998

[17] A G Freeman ldquoOptic neuropathy and chronic cyanide intox-ication a reviewrdquo Journal of the Royal Society of Medicine vol81 no 2 pp 103ndash106 1988

[18] I G Syme J Bronte-Stewart and W S Foulds ldquoClinical andbiochemical findings in Leberrsquos hereditary optic atrophyrdquoTrans-actions of the Ophthalmological Societies of the United Kingdomvol 103 no 5 pp 556ndash559 1983

[19] K Tsao P A Aitken and D R Johns ldquoSmoking as an aetio-logical factor in a pedigree with Leberrsquos hereditary optic neu-ropathyrdquo British Journal of Ophthalmology vol 83 no 5 pp577ndash581 1999

[20] S J Crews ldquoBilateral amblyopia in west Indiansrdquo Transactionsof the Ophthalmological Societies of the United Kingdom vol 83pp 653ndash667 1963

[21] A D Mackenzie and C I Phillips ldquoWest Indian amblyopiardquoBrain vol 91 no 2 pp 249ndash260 1968

[22] F D Carroll ldquoJamaican optic neuropathy in immigrants to theUnited StatesrdquoThe American Journal of Ophthalmology vol 71no 1 pp 261ndash265 1971

8 Journal of Toxicology

[23] B O Osuntokun and O Osuntokun ldquoTropical amblyopia inNigeriansrdquo American Journal of Ophthalmology vol 72 no 4pp 708ndash716 1971

[24] K Tucker and T R Hedges ldquoFood shortages and an epidemicof optic and peripheral neuropathy in CubardquoNutrition Reviewsvol 51 no 12 pp 349ndash357 1993

[25] D E McMillan and P J Geevarghese ldquoDietary cyanide andtropical malnutrition diabetesrdquo Diabetes Care vol 2 no 2 pp202ndash208 1979

[26] C S Pitchumoni N K Jain A B Lowenfels and E P DimagnoldquoChronic cyanide poisoning unifying concept for alcoholic andtropical pancreatitisrdquo Pancreas vol 3 no 2 pp 220ndash222 1988

[27] B P Kamalu ldquoPathological changes in growing dogs fed ona balanced cassava (Manihot esculenta Crantz) dietrdquo BritishJournal of Nutrition vol 69 no 3 pp 921ndash934 1993

[28] B P Kamalu ldquoThe adverse effects of long-term cassava (Mani-hot esculenta Crantz) consumptionrdquo International Journal ofFood Sciences and Nutrition vol 46 no 1 pp 65ndash93 1995

[29] B Soto-Blanco S L Gorniak and E T Kimura ldquoPhysiopatho-logical effects of the administration of chronic cyanide togrowing goatsmdasha model for ingestion of cyanogenic plantsrdquoVeterinary ResearchCommunications vol 25 no 5 pp 379ndash3892001

[30] A B Sousa B Soto-Blanco J L Guerra E T Kimura and SL Gorniak ldquoDoes prolonged oral exposure to cyanide promotehepatotoxicity and nephrotoxicityrdquo Toxicology vol 174 no 2pp 87ndash95 2002

[31] H Manzano A B de Sousa B Soto-Blanco J L Guerra P CMaiorka and S L Gorniak ldquoEffects of long-term cyanide inges-tion by pigsrdquo Veterinary Research Communications vol 31 no1 pp 93ndash104 2007

[32] J T Prichard and J L Voss ldquoFetal ankylosis in horses associatedwith hybrid Sudan pasturerdquo Journal of the American VeterinaryMedical Association vol 150 no 8 pp 871ndash873 1967

[33] L A Selby R W Menges E C Houser R E Flatt and A ACase ldquoOutbreak of swine malformations associated with thewild black cherry Prunus serotinardquo Archives of EnvironmentalHealth vol 22 no 4 pp 496ndash501 1971

[34] M G W Rodel ldquoEffects of different grasses on the incidence ofneonatal goiter and skeletal deformities in autumn born lambsrdquoRhodesia Agricultural Journal vol 69 pp 59ndash60 1972

[35] J T Seaman M G Smeal and J C Wright ldquoThe possibleassociation of a sorghum (Sorghum sudanese) hybrid as acause of developmental defects in calvesrdquo Australian VeterinaryJournal vol 57 no 7 pp 351ndash352 1981

[36] G A Bradley H C Metcalf C Reggiardo et al ldquoNeuroaxonaldegeneration in sheep grazing Sorghum pasturesrdquo Journal ofVeterinary Diagnostic Investigation vol 7 no 2 pp 229ndash2361995

[37] J D Singh ldquoThe teratogenic effects of dietary cassava on thepregnant albino rat a preliminary reportrdquo Teratology vol 24no 3 pp 289ndash291 1981

[38] H K Kumbnani A Singh G Singh and P Parimoo ldquoLimbrsquosmalformations in SouthernNigeriamdashGarri (Manihot esculenta)a probable causerdquo Journal of Human Ecology vol 1 pp 189ndash1911990

[39] B Soto-Blanco and S L Gorniak ldquoPrenatal toxicity of cyanidein goatsmdasha model for teratological studies in ruminantsrdquoTheriogenology vol 62 no 6 pp 1012ndash1026 2004

[40] A B de Sousa P CMaiorka I DGoncalves L RM de Sa andS L Gorniak ldquoEvaluation of effects of prenatal exposure to the

cyanide and thiocyanate inwistar ratsrdquoReproductive Toxicologyvol 23 no 4 pp 568ndash577 2007

[41] N C Ganderup W Harvey J T Mortensen and W HarroukldquoTheminipig as nonrodent species in toxicologymdashwhere are wenowrdquo International Journal of Toxicology vol 31 no 6 pp 507ndash528 2012

[42] G Bode P Clausing F Gervais et al ldquoThe utility of theminipigas an animal model in regulatory toxicologyrdquo Journal of Phar-macological and Toxicological Methods vol 62 no 3 pp 196ndash220 2010

[43] R D Geisert R H Renegar W W Thatcher R M Robertsand FW Bazer ldquoEstablishment of pregnancy in the pig I Inter-relationships between preimplantation development of the pigblastocyst and uterine endometrial secretionsrdquoBiology of Repro-duction vol 27 no 4 pp 925ndash939 1982

[44] K T Szabo Congenital Malformations in Laboratory and FarmAnimals Academic Press San Diego Calif USA 1989

[45] E A Iyayi ldquoDietary cyanide effect on performance and serumtestosterone of growing male pigsrdquo Beitrage zur TropischenLandwirtschaft undVeterinarmedizin vol 29 no 3 pp 347ndash3521991

[46] H Manzano A B Souza and S L Gorniak ldquoExposicaocianıdrica em suınos uma abordagem dos parametros toxic-ocineticos utilizando o tiocianato como biomarcadorrdquoBrazilianJournal of Veterinary Research and Animal Science vol 43 pp93ndash101 2006

[47] P Blanc M Hogan K Mallin D Hryhorczuk S Hessl and BBernard ldquoCyanide intoxication among silver-reclaiming work-ersrdquo The Journal of the American Medical Association vol 253no 3 pp 367ndash371 1985

[48] C F I Onwuka ldquoHydrocyanic acid contents of tropical browseand their influence on performance of goatsrdquo Food Chemistryvol 45 no 1 pp 5ndash10 1992

[49] C Ibebunjo B P Kamalu and E C Ihemelandu ldquoComparisonof the effects of cassava (Manihot esculenta Crantz) organiccyanide and inorganic cyanide on muscle and bone develop-ment in a Nigerian breed of dogrdquo British Journal of Nutritionvol 68 no 2 pp 483ndash491 1992

[50] O O Tewe and J H Maner ldquoPerformance and pathophysio-logical changes in pregnant pigs fed cassava diets containingdifferent levels of cyaniderdquo Research in Veterinary Science vol30 no 2 pp 147ndash151 1981

[51] N P Okolie and A U Osagie ldquoLiver and kidney lesions andassociated enzyme changes induced in rabbits by chronic cya-nide exposurerdquo Food and Chemical Toxicology vol 37 no 7 pp745ndash750 1999

[52] B Soto-Blanco and S L Gorniak ldquoToxic effects of prolongedadministration of leaves of cassava (Manihot esculenta Crantz)to goatsrdquo Experimental and Toxicologic Pathology vol 62 no 4pp 361ndash366 2010

[53] J Wilson ldquoCyanide in human disease a review of clinical andlaboratory evidencerdquo Fundamental and Applied Toxicology vol3 no 5 pp 397ndash399 1983

[54] E M Mills P G Gunasekar L Li J L Borowitz and G EIsom ldquoDifferential susceptibility of brain areas cyanide involvesdifferent modes of cell deathrdquo Toxicology and Applied Pharma-cology vol 156 no 1 pp 6ndash16 1999

[55] B Soto-Blanco P CesarMarioka and S Lima Gorniak ldquoEffectsof long-term low-dose cyanide administration to ratsrdquo Ecotoxi-cology and Environmental Safety vol 53 no 1 pp 37ndash41 2002

Journal of Toxicology 9

[56] B Soto-Blanco F T V Pereira A F D CarvalhoM AMiglinoand S L Gorniak ldquoFetal andmaternal lesions of cyanide dosingto pregnant goatsrdquo Small Ruminant Research vol 87 no 1ndash3 pp76ndash80 2009

[57] T Tylleskar M Banea N Bikangi G Nahimana L-A Perssonand H Rosling ldquoDietary determinants of a non-progressivespastic paraparesis (Konzo) a case-referent study in a highincidence area of Zairerdquo International Journal of Epidemiologyvol 24 no 5 pp 949ndash956 1995

[58] M M Swindle A Makin A J Herron F J Clubb Jr and K SFrazier ldquoSwine asmodels in biomedical research and toxicologytestingrdquo Veterinary Pathology vol 49 no 2 pp 344ndash356 2012

[59] B P Kamalu ldquoThe effect of a nutritionally-balanced cassava(Manihot esculenta Crantz) diet on endocrine function usingthe dog as a model 1 Pancreasrdquo British Journal of Nutrition vol65 no 3 pp 365ndash372 1991

[60] N P Okolie and A U Osagie ldquoDifferential effects of chroniccyanide intoxication on heart lung and pancreatic tissuesrdquo Foodand Chemical Toxicology vol 38 no 6 pp 543ndash548 2000

[61] M M Swindle and A C Smith ldquoComparative anatomy andphysiology of the pigrdquo Scandinavian Journal of LaboratoryAnimal Science vol 25 no 1 pp 11ndash21 1998

[62] L Jovanovic andD J Pettitt ldquoGestational diabetesmellitusrdquoTheJournal of the AmericanMedical Association vol 286 no 20 pp2516ndash2518 2001

[63] TA Buchanan andAHXiang ldquoGestational diabetesmellitusrdquoThe Journal of Clinical Investigation vol 115 no 3 pp 485ndash4912005

[64] The Practice Committee of American Society for ReproductiveMedicine ldquoSmoking and infertilityrdquo Fertility and Sterility vol4 pp 1181ndash1186 2004

[65] L Dencker R DrsquoArgy B R G Danielsson H Ghantous and GO Sperber ldquoSaturable accumulation of retinoic acid in neuraland neural crest derived cells in early embryonic developmentrdquoDevelopmental Pharmacology andTherapeutics vol 10 no 3 pp212ndash223 1987

[66] S Schwartz ldquoProviding toxicokinetic support for reproductivetoxicology studies in pharmaceutical developmentrdquo Archives ofToxicology vol 75 no 7 pp 381ndash387 2001

[67] W J Scott and H Nau ldquoAccumulation of weak acids in theyoung mammalian embryordquo in Pharmacokinetics in Teratoge-nesis H Nau and W J Scott Eds pp 72ndash76 CRC Press BocaRaton Fla USA 1987

[68] O O Tewe J H Maner and G Gomez ldquoInfluence of cassavadiets on placental thiocyanate transfer tissue rhodanese activityand performance of rats during gestationrdquo Journal of the Scienceof Food and Agriculture vol 28 no 8 pp 750ndash756 1977

[69] L Claudio C F Bearer andDWallinga ldquoAssessment of theUSenvironmental protection agency methods for identification ofhazards to developing organisms part II the developing toxicitytesting guidelinerdquo American Journal of Industrial Medicine vol35 no 6 pp 554ndash563 1999

Journal of Toxicology 7

it was not possible to conclude if ultrasound evaluation inpigs could be valuable for early detection of malformationsldquoin uterordquo

Amniocenteses in this study revealed high levels ofthiocyanate in the amniotic fluid of pregnant sows Denckeret al [65] and Schwartz [66] show that anionic substancessuch as the weak acid cyanuric acid can accumulate in theembryo and fetus This may be due to the pH gradient inmaternal plasma which is more acidic than the pH of fetalplasma Studies have thus observed an ldquoimprisonmentrdquo ofcyanide in the fetal organism [67] In addition it is knownthat the enzyme rhodanese is found in the placenta and thefetus [68] Thus it is possible that the ldquoarrestedrdquo cyanidecan be transformed into its main metabolite resulting inindirect thiocyanate toxicity to the fetus This hypothesisis additionally supported by a study in which cyanide orthiocyanate was administered to pregnant rats and bothsubstances were found to promote toxic effects in the fetuses[40]

The protocols that have been used to assess develop-mental toxicity in previous studies have certain limitations[69] One such limitation is the lack of postnatal monitoringof the development of the offspring which can result inerroneous conclusions about the developmental toxicity ofthe substances under examination In fact de Sousa et al [40]evaluated prenatal exposure to cyanide in rats and found thatalterations in pups were detected only at weaning Our studysupports these findings as we identified certain toxic effectsof cyanide in the piglets only at weaning

5 Conclusion

In conclusion pregnant sows exposed to cyanide had toxiceffects in both mothers and piglets However the toxiceffects in the offspring were observed belatedly Thereforethe data presented here reinforces the hypothesis that acomplementary evaluation of the neonate after weaning is anecessary component of studies on developmental toxicityIn addition we suggest that swine can serve as a usefulanimal model to study the neurological goitrogenic andreproductive toxicity of cyanide

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] W C Beamer R M Shealy and D S Prough ldquoAcute cyanidepoisoning from laetrile ingestionrdquo Annals of Emergency Medi-cine vol 12 no 7 pp 449ndash451 1983

[2] R Froldi M Cingolani and C Cacaci ldquoA case of suicide byingestion of sodium nitroprussiderdquo Journal of Forensic Sciencesvol 46 no 6 pp 1504ndash1506 2001

[3] J E Poulton ldquoCyanogenic compounds in plants and their toxiceffectsrdquo inHandbook of Natural Toxins Plant and Fungal ToxinsR F Keeler and A R Tu Eds vol 1 pp 117ndash157 Marcel DekkerNew York NY USA 1983

[4] United States Army Medical Research Institute of ChemicalDefense and Chemical Casualty Care Division ldquoCyaniderdquo inMedicalManagement of Chemical Causalities Handbook UnitedStates Army Medical Research Institute of Chemical DefenseAberdeen Md USA 3rd edition 2004

[5] M J Koschel ldquoWhere therersquos smoke there may be cyaniderdquoTheAmerican Journal of Nursing vol 102 no 8 pp 39ndash42 2002

[6] K Anseeuw N Delvau G Burillo-Putze et al ldquoCyanide poi-soning by fire smoke inhalation a European expert consensusrdquoEuropean Journal of Emergency Medicine vol 20 no 1 pp 2ndash92013

[7] V Schulz ldquoClinical pharmacokinetics- of nitroprusside cya-nide thiosulphate and thiocyanaterdquo Clinical Pharmacokineticsvol 9 no 3 pp 239ndash251 1984

[8] J Vetter ldquoPlant cyanogenic glycosidesrdquo Toxicon vol 38 no 1pp 11ndash36 2000

[9] B Ballantyne ldquoToxicology of cyanidesrdquo in Clinical and Exper-imental Toxicology of Cyanides B Ballantyne and T C MarrsEds pp 41ndash126 Wright Bristol UK 1987

[10] H Nzwalo and J Cliff ldquoKonzo from poverty cassava andcyanogen intake to toxico-nutritional neurological diseaserdquoPLoS Neglected Tropical Diseases vol 5 no 6 Article ID e10512011

[11] B O Osuntokun ldquoCassava diet chronic cyanide intoxicationand neuropathy in the Nigerian Africansrdquo World Review ofNutrition and Dietetics vol 36 pp 141ndash173 1981

[12] J Njoh ldquoTropical ataxic neuropathy in Liberiansrdquo Tropical andGeographical Medicine vol 42 no 1 pp 92ndash94 1990

[13] N L Rosenberg J A Myers and W R Wayne Martin ldquoCya-nide-induced parkinsonism clinical MRI and 6-fluorodopaPET studiesrdquo Neurology vol 39 no 1 pp 142ndash144 1989

[14] M Di Filippo N Tambasco G Muzi et al ldquoParkinsonism andcognitive impairment following chronic exposure to potassiumcyaniderdquoMovement Disorders vol 23 no 3 pp 468ndash470 2008

[15] A R Pettigrew and G S Fell ldquoSimplified colorimetric determi-nation of thiocyanate in biological fluids and its application toinvestigation of the toxic amblyopiasrdquo Clinical Chemistry vol18 no 9 pp 996ndash1000 1972

[16] Y Solberg M Rosner and M Belkin ldquoThe association betwencigarette smoking and ocular diseasesrdquo Survey of Ophthalmol-ogy vol 42 no 6 pp 535ndash547 1998

[17] A G Freeman ldquoOptic neuropathy and chronic cyanide intox-ication a reviewrdquo Journal of the Royal Society of Medicine vol81 no 2 pp 103ndash106 1988

[18] I G Syme J Bronte-Stewart and W S Foulds ldquoClinical andbiochemical findings in Leberrsquos hereditary optic atrophyrdquoTrans-actions of the Ophthalmological Societies of the United Kingdomvol 103 no 5 pp 556ndash559 1983

[19] K Tsao P A Aitken and D R Johns ldquoSmoking as an aetio-logical factor in a pedigree with Leberrsquos hereditary optic neu-ropathyrdquo British Journal of Ophthalmology vol 83 no 5 pp577ndash581 1999

[20] S J Crews ldquoBilateral amblyopia in west Indiansrdquo Transactionsof the Ophthalmological Societies of the United Kingdom vol 83pp 653ndash667 1963

[21] A D Mackenzie and C I Phillips ldquoWest Indian amblyopiardquoBrain vol 91 no 2 pp 249ndash260 1968

[22] F D Carroll ldquoJamaican optic neuropathy in immigrants to theUnited StatesrdquoThe American Journal of Ophthalmology vol 71no 1 pp 261ndash265 1971

8 Journal of Toxicology

[23] B O Osuntokun and O Osuntokun ldquoTropical amblyopia inNigeriansrdquo American Journal of Ophthalmology vol 72 no 4pp 708ndash716 1971

[24] K Tucker and T R Hedges ldquoFood shortages and an epidemicof optic and peripheral neuropathy in CubardquoNutrition Reviewsvol 51 no 12 pp 349ndash357 1993

[25] D E McMillan and P J Geevarghese ldquoDietary cyanide andtropical malnutrition diabetesrdquo Diabetes Care vol 2 no 2 pp202ndash208 1979

[26] C S Pitchumoni N K Jain A B Lowenfels and E P DimagnoldquoChronic cyanide poisoning unifying concept for alcoholic andtropical pancreatitisrdquo Pancreas vol 3 no 2 pp 220ndash222 1988

[27] B P Kamalu ldquoPathological changes in growing dogs fed ona balanced cassava (Manihot esculenta Crantz) dietrdquo BritishJournal of Nutrition vol 69 no 3 pp 921ndash934 1993

[28] B P Kamalu ldquoThe adverse effects of long-term cassava (Mani-hot esculenta Crantz) consumptionrdquo International Journal ofFood Sciences and Nutrition vol 46 no 1 pp 65ndash93 1995

[29] B Soto-Blanco S L Gorniak and E T Kimura ldquoPhysiopatho-logical effects of the administration of chronic cyanide togrowing goatsmdasha model for ingestion of cyanogenic plantsrdquoVeterinary ResearchCommunications vol 25 no 5 pp 379ndash3892001

[30] A B Sousa B Soto-Blanco J L Guerra E T Kimura and SL Gorniak ldquoDoes prolonged oral exposure to cyanide promotehepatotoxicity and nephrotoxicityrdquo Toxicology vol 174 no 2pp 87ndash95 2002

[31] H Manzano A B de Sousa B Soto-Blanco J L Guerra P CMaiorka and S L Gorniak ldquoEffects of long-term cyanide inges-tion by pigsrdquo Veterinary Research Communications vol 31 no1 pp 93ndash104 2007

[32] J T Prichard and J L Voss ldquoFetal ankylosis in horses associatedwith hybrid Sudan pasturerdquo Journal of the American VeterinaryMedical Association vol 150 no 8 pp 871ndash873 1967

[33] L A Selby R W Menges E C Houser R E Flatt and A ACase ldquoOutbreak of swine malformations associated with thewild black cherry Prunus serotinardquo Archives of EnvironmentalHealth vol 22 no 4 pp 496ndash501 1971

[34] M G W Rodel ldquoEffects of different grasses on the incidence ofneonatal goiter and skeletal deformities in autumn born lambsrdquoRhodesia Agricultural Journal vol 69 pp 59ndash60 1972

[35] J T Seaman M G Smeal and J C Wright ldquoThe possibleassociation of a sorghum (Sorghum sudanese) hybrid as acause of developmental defects in calvesrdquo Australian VeterinaryJournal vol 57 no 7 pp 351ndash352 1981

[36] G A Bradley H C Metcalf C Reggiardo et al ldquoNeuroaxonaldegeneration in sheep grazing Sorghum pasturesrdquo Journal ofVeterinary Diagnostic Investigation vol 7 no 2 pp 229ndash2361995

[37] J D Singh ldquoThe teratogenic effects of dietary cassava on thepregnant albino rat a preliminary reportrdquo Teratology vol 24no 3 pp 289ndash291 1981

[38] H K Kumbnani A Singh G Singh and P Parimoo ldquoLimbrsquosmalformations in SouthernNigeriamdashGarri (Manihot esculenta)a probable causerdquo Journal of Human Ecology vol 1 pp 189ndash1911990

[39] B Soto-Blanco and S L Gorniak ldquoPrenatal toxicity of cyanidein goatsmdasha model for teratological studies in ruminantsrdquoTheriogenology vol 62 no 6 pp 1012ndash1026 2004

[40] A B de Sousa P CMaiorka I DGoncalves L RM de Sa andS L Gorniak ldquoEvaluation of effects of prenatal exposure to the

cyanide and thiocyanate inwistar ratsrdquoReproductive Toxicologyvol 23 no 4 pp 568ndash577 2007

[41] N C Ganderup W Harvey J T Mortensen and W HarroukldquoTheminipig as nonrodent species in toxicologymdashwhere are wenowrdquo International Journal of Toxicology vol 31 no 6 pp 507ndash528 2012

[42] G Bode P Clausing F Gervais et al ldquoThe utility of theminipigas an animal model in regulatory toxicologyrdquo Journal of Phar-macological and Toxicological Methods vol 62 no 3 pp 196ndash220 2010

[43] R D Geisert R H Renegar W W Thatcher R M Robertsand FW Bazer ldquoEstablishment of pregnancy in the pig I Inter-relationships between preimplantation development of the pigblastocyst and uterine endometrial secretionsrdquoBiology of Repro-duction vol 27 no 4 pp 925ndash939 1982

[44] K T Szabo Congenital Malformations in Laboratory and FarmAnimals Academic Press San Diego Calif USA 1989

[45] E A Iyayi ldquoDietary cyanide effect on performance and serumtestosterone of growing male pigsrdquo Beitrage zur TropischenLandwirtschaft undVeterinarmedizin vol 29 no 3 pp 347ndash3521991

[46] H Manzano A B Souza and S L Gorniak ldquoExposicaocianıdrica em suınos uma abordagem dos parametros toxic-ocineticos utilizando o tiocianato como biomarcadorrdquoBrazilianJournal of Veterinary Research and Animal Science vol 43 pp93ndash101 2006

[47] P Blanc M Hogan K Mallin D Hryhorczuk S Hessl and BBernard ldquoCyanide intoxication among silver-reclaiming work-ersrdquo The Journal of the American Medical Association vol 253no 3 pp 367ndash371 1985

[48] C F I Onwuka ldquoHydrocyanic acid contents of tropical browseand their influence on performance of goatsrdquo Food Chemistryvol 45 no 1 pp 5ndash10 1992

[49] C Ibebunjo B P Kamalu and E C Ihemelandu ldquoComparisonof the effects of cassava (Manihot esculenta Crantz) organiccyanide and inorganic cyanide on muscle and bone develop-ment in a Nigerian breed of dogrdquo British Journal of Nutritionvol 68 no 2 pp 483ndash491 1992

[50] O O Tewe and J H Maner ldquoPerformance and pathophysio-logical changes in pregnant pigs fed cassava diets containingdifferent levels of cyaniderdquo Research in Veterinary Science vol30 no 2 pp 147ndash151 1981

[51] N P Okolie and A U Osagie ldquoLiver and kidney lesions andassociated enzyme changes induced in rabbits by chronic cya-nide exposurerdquo Food and Chemical Toxicology vol 37 no 7 pp745ndash750 1999

[52] B Soto-Blanco and S L Gorniak ldquoToxic effects of prolongedadministration of leaves of cassava (Manihot esculenta Crantz)to goatsrdquo Experimental and Toxicologic Pathology vol 62 no 4pp 361ndash366 2010

[53] J Wilson ldquoCyanide in human disease a review of clinical andlaboratory evidencerdquo Fundamental and Applied Toxicology vol3 no 5 pp 397ndash399 1983

[54] E M Mills P G Gunasekar L Li J L Borowitz and G EIsom ldquoDifferential susceptibility of brain areas cyanide involvesdifferent modes of cell deathrdquo Toxicology and Applied Pharma-cology vol 156 no 1 pp 6ndash16 1999

[55] B Soto-Blanco P CesarMarioka and S Lima Gorniak ldquoEffectsof long-term low-dose cyanide administration to ratsrdquo Ecotoxi-cology and Environmental Safety vol 53 no 1 pp 37ndash41 2002

Journal of Toxicology 9

[56] B Soto-Blanco F T V Pereira A F D CarvalhoM AMiglinoand S L Gorniak ldquoFetal andmaternal lesions of cyanide dosingto pregnant goatsrdquo Small Ruminant Research vol 87 no 1ndash3 pp76ndash80 2009

[57] T Tylleskar M Banea N Bikangi G Nahimana L-A Perssonand H Rosling ldquoDietary determinants of a non-progressivespastic paraparesis (Konzo) a case-referent study in a highincidence area of Zairerdquo International Journal of Epidemiologyvol 24 no 5 pp 949ndash956 1995

[58] M M Swindle A Makin A J Herron F J Clubb Jr and K SFrazier ldquoSwine asmodels in biomedical research and toxicologytestingrdquo Veterinary Pathology vol 49 no 2 pp 344ndash356 2012

[59] B P Kamalu ldquoThe effect of a nutritionally-balanced cassava(Manihot esculenta Crantz) diet on endocrine function usingthe dog as a model 1 Pancreasrdquo British Journal of Nutrition vol65 no 3 pp 365ndash372 1991

[60] N P Okolie and A U Osagie ldquoDifferential effects of chroniccyanide intoxication on heart lung and pancreatic tissuesrdquo Foodand Chemical Toxicology vol 38 no 6 pp 543ndash548 2000

[61] M M Swindle and A C Smith ldquoComparative anatomy andphysiology of the pigrdquo Scandinavian Journal of LaboratoryAnimal Science vol 25 no 1 pp 11ndash21 1998

[62] L Jovanovic andD J Pettitt ldquoGestational diabetesmellitusrdquoTheJournal of the AmericanMedical Association vol 286 no 20 pp2516ndash2518 2001

[63] TA Buchanan andAHXiang ldquoGestational diabetesmellitusrdquoThe Journal of Clinical Investigation vol 115 no 3 pp 485ndash4912005

[64] The Practice Committee of American Society for ReproductiveMedicine ldquoSmoking and infertilityrdquo Fertility and Sterility vol4 pp 1181ndash1186 2004

[65] L Dencker R DrsquoArgy B R G Danielsson H Ghantous and GO Sperber ldquoSaturable accumulation of retinoic acid in neuraland neural crest derived cells in early embryonic developmentrdquoDevelopmental Pharmacology andTherapeutics vol 10 no 3 pp212ndash223 1987

[66] S Schwartz ldquoProviding toxicokinetic support for reproductivetoxicology studies in pharmaceutical developmentrdquo Archives ofToxicology vol 75 no 7 pp 381ndash387 2001

[67] W J Scott and H Nau ldquoAccumulation of weak acids in theyoung mammalian embryordquo in Pharmacokinetics in Teratoge-nesis H Nau and W J Scott Eds pp 72ndash76 CRC Press BocaRaton Fla USA 1987

[68] O O Tewe J H Maner and G Gomez ldquoInfluence of cassavadiets on placental thiocyanate transfer tissue rhodanese activityand performance of rats during gestationrdquo Journal of the Scienceof Food and Agriculture vol 28 no 8 pp 750ndash756 1977

[69] L Claudio C F Bearer andDWallinga ldquoAssessment of theUSenvironmental protection agency methods for identification ofhazards to developing organisms part II the developing toxicitytesting guidelinerdquo American Journal of Industrial Medicine vol35 no 6 pp 554ndash563 1999

8 Journal of Toxicology

[23] B O Osuntokun and O Osuntokun ldquoTropical amblyopia inNigeriansrdquo American Journal of Ophthalmology vol 72 no 4pp 708ndash716 1971

[24] K Tucker and T R Hedges ldquoFood shortages and an epidemicof optic and peripheral neuropathy in CubardquoNutrition Reviewsvol 51 no 12 pp 349ndash357 1993

[25] D E McMillan and P J Geevarghese ldquoDietary cyanide andtropical malnutrition diabetesrdquo Diabetes Care vol 2 no 2 pp202ndash208 1979

[26] C S Pitchumoni N K Jain A B Lowenfels and E P DimagnoldquoChronic cyanide poisoning unifying concept for alcoholic andtropical pancreatitisrdquo Pancreas vol 3 no 2 pp 220ndash222 1988

[27] B P Kamalu ldquoPathological changes in growing dogs fed ona balanced cassava (Manihot esculenta Crantz) dietrdquo BritishJournal of Nutrition vol 69 no 3 pp 921ndash934 1993

[28] B P Kamalu ldquoThe adverse effects of long-term cassava (Mani-hot esculenta Crantz) consumptionrdquo International Journal ofFood Sciences and Nutrition vol 46 no 1 pp 65ndash93 1995

[29] B Soto-Blanco S L Gorniak and E T Kimura ldquoPhysiopatho-logical effects of the administration of chronic cyanide togrowing goatsmdasha model for ingestion of cyanogenic plantsrdquoVeterinary ResearchCommunications vol 25 no 5 pp 379ndash3892001

[30] A B Sousa B Soto-Blanco J L Guerra E T Kimura and SL Gorniak ldquoDoes prolonged oral exposure to cyanide promotehepatotoxicity and nephrotoxicityrdquo Toxicology vol 174 no 2pp 87ndash95 2002

[31] H Manzano A B de Sousa B Soto-Blanco J L Guerra P CMaiorka and S L Gorniak ldquoEffects of long-term cyanide inges-tion by pigsrdquo Veterinary Research Communications vol 31 no1 pp 93ndash104 2007

[32] J T Prichard and J L Voss ldquoFetal ankylosis in horses associatedwith hybrid Sudan pasturerdquo Journal of the American VeterinaryMedical Association vol 150 no 8 pp 871ndash873 1967

[33] L A Selby R W Menges E C Houser R E Flatt and A ACase ldquoOutbreak of swine malformations associated with thewild black cherry Prunus serotinardquo Archives of EnvironmentalHealth vol 22 no 4 pp 496ndash501 1971

[34] M G W Rodel ldquoEffects of different grasses on the incidence ofneonatal goiter and skeletal deformities in autumn born lambsrdquoRhodesia Agricultural Journal vol 69 pp 59ndash60 1972

[35] J T Seaman M G Smeal and J C Wright ldquoThe possibleassociation of a sorghum (Sorghum sudanese) hybrid as acause of developmental defects in calvesrdquo Australian VeterinaryJournal vol 57 no 7 pp 351ndash352 1981

[36] G A Bradley H C Metcalf C Reggiardo et al ldquoNeuroaxonaldegeneration in sheep grazing Sorghum pasturesrdquo Journal ofVeterinary Diagnostic Investigation vol 7 no 2 pp 229ndash2361995

[37] J D Singh ldquoThe teratogenic effects of dietary cassava on thepregnant albino rat a preliminary reportrdquo Teratology vol 24no 3 pp 289ndash291 1981

[38] H K Kumbnani A Singh G Singh and P Parimoo ldquoLimbrsquosmalformations in SouthernNigeriamdashGarri (Manihot esculenta)a probable causerdquo Journal of Human Ecology vol 1 pp 189ndash1911990

[39] B Soto-Blanco and S L Gorniak ldquoPrenatal toxicity of cyanidein goatsmdasha model for teratological studies in ruminantsrdquoTheriogenology vol 62 no 6 pp 1012ndash1026 2004

[40] A B de Sousa P CMaiorka I DGoncalves L RM de Sa andS L Gorniak ldquoEvaluation of effects of prenatal exposure to the

cyanide and thiocyanate inwistar ratsrdquoReproductive Toxicologyvol 23 no 4 pp 568ndash577 2007

[41] N C Ganderup W Harvey J T Mortensen and W HarroukldquoTheminipig as nonrodent species in toxicologymdashwhere are wenowrdquo International Journal of Toxicology vol 31 no 6 pp 507ndash528 2012

[42] G Bode P Clausing F Gervais et al ldquoThe utility of theminipigas an animal model in regulatory toxicologyrdquo Journal of Phar-macological and Toxicological Methods vol 62 no 3 pp 196ndash220 2010

[43] R D Geisert R H Renegar W W Thatcher R M Robertsand FW Bazer ldquoEstablishment of pregnancy in the pig I Inter-relationships between preimplantation development of the pigblastocyst and uterine endometrial secretionsrdquoBiology of Repro-duction vol 27 no 4 pp 925ndash939 1982

[44] K T Szabo Congenital Malformations in Laboratory and FarmAnimals Academic Press San Diego Calif USA 1989

[45] E A Iyayi ldquoDietary cyanide effect on performance and serumtestosterone of growing male pigsrdquo Beitrage zur TropischenLandwirtschaft undVeterinarmedizin vol 29 no 3 pp 347ndash3521991

[46] H Manzano A B Souza and S L Gorniak ldquoExposicaocianıdrica em suınos uma abordagem dos parametros toxic-ocineticos utilizando o tiocianato como biomarcadorrdquoBrazilianJournal of Veterinary Research and Animal Science vol 43 pp93ndash101 2006

[47] P Blanc M Hogan K Mallin D Hryhorczuk S Hessl and BBernard ldquoCyanide intoxication among silver-reclaiming work-ersrdquo The Journal of the American Medical Association vol 253no 3 pp 367ndash371 1985

[48] C F I Onwuka ldquoHydrocyanic acid contents of tropical browseand their influence on performance of goatsrdquo Food Chemistryvol 45 no 1 pp 5ndash10 1992

[49] C Ibebunjo B P Kamalu and E C Ihemelandu ldquoComparisonof the effects of cassava (Manihot esculenta Crantz) organiccyanide and inorganic cyanide on muscle and bone develop-ment in a Nigerian breed of dogrdquo British Journal of Nutritionvol 68 no 2 pp 483ndash491 1992

[50] O O Tewe and J H Maner ldquoPerformance and pathophysio-logical changes in pregnant pigs fed cassava diets containingdifferent levels of cyaniderdquo Research in Veterinary Science vol30 no 2 pp 147ndash151 1981

[51] N P Okolie and A U Osagie ldquoLiver and kidney lesions andassociated enzyme changes induced in rabbits by chronic cya-nide exposurerdquo Food and Chemical Toxicology vol 37 no 7 pp745ndash750 1999

[52] B Soto-Blanco and S L Gorniak ldquoToxic effects of prolongedadministration of leaves of cassava (Manihot esculenta Crantz)to goatsrdquo Experimental and Toxicologic Pathology vol 62 no 4pp 361ndash366 2010

[53] J Wilson ldquoCyanide in human disease a review of clinical andlaboratory evidencerdquo Fundamental and Applied Toxicology vol3 no 5 pp 397ndash399 1983

[54] E M Mills P G Gunasekar L Li J L Borowitz and G EIsom ldquoDifferential susceptibility of brain areas cyanide involvesdifferent modes of cell deathrdquo Toxicology and Applied Pharma-cology vol 156 no 1 pp 6ndash16 1999

[55] B Soto-Blanco P CesarMarioka and S Lima Gorniak ldquoEffectsof long-term low-dose cyanide administration to ratsrdquo Ecotoxi-cology and Environmental Safety vol 53 no 1 pp 37ndash41 2002

Journal of Toxicology 9

[56] B Soto-Blanco F T V Pereira A F D CarvalhoM AMiglinoand S L Gorniak ldquoFetal andmaternal lesions of cyanide dosingto pregnant goatsrdquo Small Ruminant Research vol 87 no 1ndash3 pp76ndash80 2009

[57] T Tylleskar M Banea N Bikangi G Nahimana L-A Perssonand H Rosling ldquoDietary determinants of a non-progressivespastic paraparesis (Konzo) a case-referent study in a highincidence area of Zairerdquo International Journal of Epidemiologyvol 24 no 5 pp 949ndash956 1995

[58] M M Swindle A Makin A J Herron F J Clubb Jr and K SFrazier ldquoSwine asmodels in biomedical research and toxicologytestingrdquo Veterinary Pathology vol 49 no 2 pp 344ndash356 2012

[59] B P Kamalu ldquoThe effect of a nutritionally-balanced cassava(Manihot esculenta Crantz) diet on endocrine function usingthe dog as a model 1 Pancreasrdquo British Journal of Nutrition vol65 no 3 pp 365ndash372 1991

[60] N P Okolie and A U Osagie ldquoDifferential effects of chroniccyanide intoxication on heart lung and pancreatic tissuesrdquo Foodand Chemical Toxicology vol 38 no 6 pp 543ndash548 2000

[61] M M Swindle and A C Smith ldquoComparative anatomy andphysiology of the pigrdquo Scandinavian Journal of LaboratoryAnimal Science vol 25 no 1 pp 11ndash21 1998

[62] L Jovanovic andD J Pettitt ldquoGestational diabetesmellitusrdquoTheJournal of the AmericanMedical Association vol 286 no 20 pp2516ndash2518 2001

[63] TA Buchanan andAHXiang ldquoGestational diabetesmellitusrdquoThe Journal of Clinical Investigation vol 115 no 3 pp 485ndash4912005

[64] The Practice Committee of American Society for ReproductiveMedicine ldquoSmoking and infertilityrdquo Fertility and Sterility vol4 pp 1181ndash1186 2004

[65] L Dencker R DrsquoArgy B R G Danielsson H Ghantous and GO Sperber ldquoSaturable accumulation of retinoic acid in neuraland neural crest derived cells in early embryonic developmentrdquoDevelopmental Pharmacology andTherapeutics vol 10 no 3 pp212ndash223 1987

[66] S Schwartz ldquoProviding toxicokinetic support for reproductivetoxicology studies in pharmaceutical developmentrdquo Archives ofToxicology vol 75 no 7 pp 381ndash387 2001

[67] W J Scott and H Nau ldquoAccumulation of weak acids in theyoung mammalian embryordquo in Pharmacokinetics in Teratoge-nesis H Nau and W J Scott Eds pp 72ndash76 CRC Press BocaRaton Fla USA 1987

[68] O O Tewe J H Maner and G Gomez ldquoInfluence of cassavadiets on placental thiocyanate transfer tissue rhodanese activityand performance of rats during gestationrdquo Journal of the Scienceof Food and Agriculture vol 28 no 8 pp 750ndash756 1977

[69] L Claudio C F Bearer andDWallinga ldquoAssessment of theUSenvironmental protection agency methods for identification ofhazards to developing organisms part II the developing toxicitytesting guidelinerdquo American Journal of Industrial Medicine vol35 no 6 pp 554ndash563 1999

Journal of Toxicology 9

[56] B Soto-Blanco F T V Pereira A F D CarvalhoM AMiglinoand S L Gorniak ldquoFetal andmaternal lesions of cyanide dosingto pregnant goatsrdquo Small Ruminant Research vol 87 no 1ndash3 pp76ndash80 2009

[57] T Tylleskar M Banea N Bikangi G Nahimana L-A Perssonand H Rosling ldquoDietary determinants of a non-progressivespastic paraparesis (Konzo) a case-referent study in a highincidence area of Zairerdquo International Journal of Epidemiologyvol 24 no 5 pp 949ndash956 1995

[58] M M Swindle A Makin A J Herron F J Clubb Jr and K SFrazier ldquoSwine asmodels in biomedical research and toxicologytestingrdquo Veterinary Pathology vol 49 no 2 pp 344ndash356 2012

[59] B P Kamalu ldquoThe effect of a nutritionally-balanced cassava(Manihot esculenta Crantz) diet on endocrine function usingthe dog as a model 1 Pancreasrdquo British Journal of Nutrition vol65 no 3 pp 365ndash372 1991

[60] N P Okolie and A U Osagie ldquoDifferential effects of chroniccyanide intoxication on heart lung and pancreatic tissuesrdquo Foodand Chemical Toxicology vol 38 no 6 pp 543ndash548 2000

[61] M M Swindle and A C Smith ldquoComparative anatomy andphysiology of the pigrdquo Scandinavian Journal of LaboratoryAnimal Science vol 25 no 1 pp 11ndash21 1998

[62] L Jovanovic andD J Pettitt ldquoGestational diabetesmellitusrdquoTheJournal of the AmericanMedical Association vol 286 no 20 pp2516ndash2518 2001

[63] TA Buchanan andAHXiang ldquoGestational diabetesmellitusrdquoThe Journal of Clinical Investigation vol 115 no 3 pp 485ndash4912005

[64] The Practice Committee of American Society for ReproductiveMedicine ldquoSmoking and infertilityrdquo Fertility and Sterility vol4 pp 1181ndash1186 2004

[65] L Dencker R DrsquoArgy B R G Danielsson H Ghantous and GO Sperber ldquoSaturable accumulation of retinoic acid in neuraland neural crest derived cells in early embryonic developmentrdquoDevelopmental Pharmacology andTherapeutics vol 10 no 3 pp212ndash223 1987

[66] S Schwartz ldquoProviding toxicokinetic support for reproductivetoxicology studies in pharmaceutical developmentrdquo Archives ofToxicology vol 75 no 7 pp 381ndash387 2001

[67] W J Scott and H Nau ldquoAccumulation of weak acids in theyoung mammalian embryordquo in Pharmacokinetics in Teratoge-nesis H Nau and W J Scott Eds pp 72ndash76 CRC Press BocaRaton Fla USA 1987

[68] O O Tewe J H Maner and G Gomez ldquoInfluence of cassavadiets on placental thiocyanate transfer tissue rhodanese activityand performance of rats during gestationrdquo Journal of the Scienceof Food and Agriculture vol 28 no 8 pp 750ndash756 1977

[69] L Claudio C F Bearer andDWallinga ldquoAssessment of theUSenvironmental protection agency methods for identification ofhazards to developing organisms part II the developing toxicitytesting guidelinerdquo American Journal of Industrial Medicine vol35 no 6 pp 554ndash563 1999