Carryover of cadmium from feed in growing pigs

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Carryover of cadmium from feed in growing pigsRon L.A.P. Hoogenbooma, Jasper Hattinkb, Ab van Polanena, Sjaak van Oostroma, John T.Verbunta, Wim A. Traaga, Kees A. Kanb, Jan C.H. van Eijkerenc, Gudrun De Boeckd & MarcoJ. Zeilmakerc

a RIKILT Institute of Food Safety, Wageningen UR, Wageningen, the Netherlandsb Animal Sciences Group, Wageningen UR, Lelystad, the Netherlandsc RIVM, National Institute of Public Health and the Environment, Bilthoven, the Netherlandsd Department of Biology, University of Antwerp, Systemic Physiological and EcotoxicologicalResearch (SPHERE), Antwerp, BelgiumPublished online: 01 Dec 2014.

To cite this article: Ron L.A.P. Hoogenboom, Jasper Hattink, Ab van Polanen, Sjaak van Oostrom, John T. Verbunt, Wim A.Traag, Kees A. Kan, Jan C.H. van Eijkeren, Gudrun De Boeck & Marco J. Zeilmaker (2014): Carryover of cadmium from feed ingrowing pigs, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2014.979370

To link to this article: http://dx.doi.org/10.1080/19440049.2014.979370

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Carryover of cadmium from feed in growing pigs

Ron L.A.P. Hoogenbooma*, Jasper Hattinkb, Ab van Polanena, Sjaak van Oostroma, John T. Verbunta, Wim A. Traaga,Kees A. Kanb, Jan C.H. van Eijkerenc, Gudrun De Boeckd and Marco J. Zeilmakerc

aRIKILT Institute of Food Safety, Wageningen UR, Wageningen, the Netherlands; bAnimal Sciences Group, Wageningen UR, Lelystad,the Netherlands; cRIVM, National Institute of Public Health and the Environment, Bilthoven, the Netherlands; dDepartment of Biology,University of Antwerp, Systemic Physiological and Ecotoxicological Research (SPHERE), Antwerp, Belgium

(Received 7 August 2014; accepted 18 October 2014)

Growing male pigs were exposed to cadmium (Cd) at levels around 1 and 10 mg kg–1 feed for up to 12 weeks, administered asCdCl2 or Cd-cysteine (CdCys). Pigs exposed to 10 mg kg–1 showed decreased growth during the last 3 weeks. Liver andkidney concentrations of Cd continuously increased over the entire 12-week exposure, exceeding the European Union limitsof 1.0 mg kg–1 (kidney) and 0.5 mg kg–1 (liver) within 3 weeks at the feed level of 10 mg kg–1. A switch to clean feed after3 weeks for 5 or 9 weeks resulted in steadily decreased levels in kidney and liver, which could be completely attributed toorgan growth. At the lower feed level, the level in kidney exceeded the limit almost twofold after 12 weeks, but not after3 weeks. Liver levels remained below the limit. Metallothionein (MT) levels in livers showed a steady decrease in bothuntreated and treated animals over time. In kidney such a decrease was only observed in control animals, whereas in thehighest-dosed animals the MT concentrations steadily increased. The observed carryover of Cd from feed to liver and kidneywas modelled by means of a simple transfer model relating levels in feed via MT levels to accumulation of Cd. Using thismodel, it was shown that the exposure period of growing pigs to feed containing the European Union limit of 0.5 mg kg–1 feedshould be less than 12 weeks in order to prevent Cd levels in the kidneys to exceed the European Union limit.

Keywords: pigs; cadmium; transfer model; metallothioneins; organs

Introduction

Heavy metals like lead, mercury and cadmium (Cd) maypresent a human health risk following the consumption ofanimal-derived products that are contaminated due to thecarryover from contaminated feed. In the case of Cd, thetoxic risk is aggravated by its accumulation in the targetorgans kidney and liver. Cd may occur as a contaminant inminerals used as feed ingredients (Sapunar-Postružniket al. 2001), but also be present in plants grown on soilof contaminated areas, like in the Kempen in Belgium andthe Netherlands due to the production of zinc (Franz et al.2008). Also the application of contaminated sewage (Lisket al. 1982; Bache et al. 1987) or manure (Linden et al.1999) may lead to elevated levels. In addition, the issuemay be relevant due to the intake of contaminated soil byanimals. To ensure the safety of consumers, maximumlimits have been set by the European Union for bothfood and feed with, for example, limits for pig feedbeing 0.5 mg kg–1 (EU 2002) and 1.0, 0.5 and 0.05 mg kg–1

for pig kidney, liver and meat respectively (EU 2006).In order to guarantee that limits for feed are low

enough to ensure compliance of various products offood-producing animals, it is important to understandthe relationship between levels in feed and food. As

shown previously for dioxins and PCBs, and aflatoxinB1 (Van Eijkeren et al. 2006a, 2006b; Hoogenboomet al. 2007, 2010), such knowledge may not only helpin tuning feed to food limits, but also be important forevaluating the potential risks during an incident and forcalculating the duration of the period on clean feed toreturn to compliant levels in food. The aim of thepresent study was to study the transfer of Cd in grow-ing pigs and to model this transfer to the organs ofinterest, i.e. liver and kidney. Though various studieshave been performed with Cd in growing pigs (Bacheet al. 1987; King et al. 1992; Zacharias et al. 2001),none of these studies included different time points ofexposure and the effect of changing the contaminatedfeed by clean feed, information required for propermodelling of the data. To overcome this deficiency, astudy on the kinetics of Cd was performed, includingthe levels in various organs and blood. As metallothio-neins (MTs) play an essential role in the accumulationof Cd in tissues like liver and kidney (Margoshes et al.1957; Kägi & Vallee 1960; Henry et al. 1994), levels ofthese proteins were determined in these organs. Theexperimental data were used to develop a mechanisti-cally based transfer model for Cd to these organs.

*Corresponding author. Email: ron.hoogenboom@wur.nlPresent address of Jasper Hattink: Environmental Scientifics Group Ltd, ESG House, Bretby Business Park, Burton upon Trent DE150YZ, UK

Food Additives & Contaminants: Part A, 2014http://dx.doi.org/10.1080/19440049.2014.979370

Another important fact is the speciation of Cd in feed.For example, when plants are grown on contaminated soil,part of the Cd may be converted to organic forms thatupon ingestion by animals may show different kineticsthan the inorganic form. For this reason, Cd-cysteine wasprepared and next to CdCl2 used to expose the pigs.

Materials and methods

Materials

Cadmium chloride (CdCl2) was purchased from Merck(Darmstadt, Germany). Cadmium-cysteine (CdCys) wasprepared as described by Cherian (1979) by mixing asolution of cysteine and CdCl2 and adjusting the pH to 8with NaOH. At this pH, the CdCys precipitated and wascollected by centrifugation. To increase the yield, CdCl2was added to the supernatant until the remaining cysteineprecipitated as well. The CdCys was lyophilised and theCd content determined with electrothermal atomic absorp-tion spectrometry (ETAAS).

Feeds were prepared by Research Diet Services (Wijkbij Duurstede, the Netherlands), both starter and growerfeeds, without or with either CdCl2 or CdCys. Feed com-position (in g kg–1) was as follows: barley 347.8, wheat200.0, peas 50.0, soybean meal 150.0, wheat semolina60.0, rapeseed meal 50.0, sugarcane molasses 20.0, potatoprotein 25.0, whey powder 40.0, soy oil 34.5, chalk 13.3,premix ID for growth 2.0, di-calcium phosphate 4.5, NaCl2.5, lysine HCl 0.2 and DL-methionine 0.2. Cd was addedas CdCl2 or CdCys and Cd levels were aimed at 1 and10 mg kg–1. Levels were checked by analysis (seeResults).

Animal study

The animal study was carried out at ASG in Lelystad. Thestudy was reviewed and approved by the EthicalCommittee. Three-month-old male pigs were divided invarious treatment groups, as shown in Figure 1:

● Group 1 was the control group and received noadditional Cd. Before starting the treatment, threeanimals were slaughtered (time 0) and subsequentlyat 3 and 12 weeks (both three animals). After3 weeks the starter feed was replaced by growerfeed.

● Groups 2 and 3, each consisting of 12 animals,received feed with respectively 1 or 10 mg Cd, asCdCl2, per kg feed for a period of 3 weeks. At thattime point three animals per group were slaugh-tered. Another three pigs of each group wereswitched to grower feeds with the same levels ofCdCl2 for another 9 weeks. The remaining six ani-mals were put on clean grower feed in order to

mimic a short-term incident with contaminatedfeed. From these six animals in each group, threewere slaughtered after 5 weeks and three after9 weeks.

● Two additional groups (4 and 5) of each 12 animalsreceived CdCys at an intended Cd level of 1 and10 mg kg–1 feed for 3 weeks and were subsequentlyswitched to grower feeds with similar contamina-tion for another 9 weeks or put on clean grower feedfor 5 or 9 weeks.

Animals were housed in groups of three. Feed was sup-plied ad libitum. During the first 3 weeks the starter feedwas supplied on specific days, starting with 44 kg on day1 and adding 20 kg on days 7, 13 and 17, and a varyingamount on day 18 depending on the consumption thus far.On day 22 the remains were weighed to determine theoverall feed consumption per cage of three animals. Atday 22 a portion of 44 kg grower feed was suppliedfollowed by 20 kg every 3 days. On day 57 the remainswere weighed and the overall consumption determined forthe 3–8-week period. On day 57, per cage, 50 kg weresupplied followed by 20 kg every second or third day.

Figure 1. Set-up of the animal study.

2 R.L.A.P. Hoogenboom et al.

Remains were determined on day 84 to calculate the feedconsumption over the period of 8–12 weeks. Animalswere weighed at day 0 and after 3, 8 and 12 weeks,when part of the animals were slaughtered.

Blood samples were collected from the animals kepton contaminated feed for 12 weeks on days 1, 3, 7, 14, 21,28, 35, 42, 49, 56, 63, 70 and 84. Animals transferred toclean feed after 3 weeks were sampled on days 1, 3, 7, 14,21, 22, 24, 28, 35, 42, 49, 56, 63, 70 and 84.

Animals were slaughtered after 0, 3, 8 and 12 weeks.Various tissues were sampled, including liver, kidney,muscle, duodenum, ileum, jejunum, lung, brain, testicleand bone.

Analysis of cadmium

Samples were pre-treated using acid digestion with amicrowave oven (MARS 5). In short, 1 g of feed or 2 gof liver, kidney, muscle, other tissue or blood were mixedwith 10 ml 70% HNO3 and heated in a microwave oven to210°C.

Acid digests were analysed using an ETAAS withZeeman background correction, using a graphite-coatedfurnace and graphite tube to vaporise the sample. Cdconcentrations were determined from a working curve(linear) after calibrating the instrument with standards ofknown concentration. The Cd concentrations were mea-sured at 228.8 nm.

The validation for liver, kidney, muscle, blood andother tissues was performed for five levels between 0.05and 1.5 mg Cd kg–1. The recovery in that range wasbetween 91% and 106%, the repeatability between 12%and 23% and the within-laboratory reproducibilitybetween 13% and 32%. The accuracy of the referencematerial (1566b ‘Oyster Tissue, National Institute ofStandards & Technology, with a certified mass fractionvalue for Cd of 2.48 ± 0.08 mg kg–1) was 98%. Theexpanded measurement uncertainty with a coverage factorof 2 was between 9% and 22%. The LOD and LOQ formeat and tissues were 0.05 and 0.1 mg kg–1 respectively.For blood the LOD was lowered to 0.001 mg kg–1 byincreasing the weight and the injection volume in theETAAS.

For feed the validation was performed in the rangefrom 0.02 to 4.5 mg kg–1. The accuracy of the referencematerial (Certified Reference Material BCR – 279, SeaLettuce, Institute for Reference Materials andMeasurements; certified mass fraction for Cd based ondry mass of 0.274 ± 0.022 mg kg–1) was 96%, the repeat-ability 16%, and the within-laboratory reproducibility33%. The expanded measurement uncertainty with a cov-erage factor of 2 was 24%. LOD and LOQ were 0.004 and0.007 mg kg–1 respectively.

RIKILT routinely participates in international ringtests, such as those from the EURL (Joint Research

Centre in Geel) and FAPAS (Food Analysis PerformanceAssessment Scheme, York). The z-scores were between –1.0 and 1.0 over the years.

Analysis of metallothioneins

For the determination of MTs, kidneys and livers werepartially thawed overnight in a cooled chamber and homo-genised as a whole in a commercial food blender. Asubsample of approximately 1 g was subsequently homo-genised on ice with 4 vols of buffer A (10 mM tris-HCl,86 mM NaCl, pH 7.4) at 4°C with an Ultra-turrax T3Homonizer (IKA, Labor technique, Shaufer, Germany).Tissue and cell homogenates were centrifuged at16 000g at 4°C in 20 min and supernatant fractions weretaken and kept at –80°C for further processing.

Cytosols of the tissues were analysed following theprocedure described by Klein et al. (1994). This methodallows the quantification of total MT in biological materi-als, including the oxidised and aggregated MT, since par-ticularly Cu-containing MT has the tendency topolymerise. This was a modification of an earlierdescribed procedure specially developed to quantify Cu-containing MT (Klein et al. 1990). The main features ofthe procedure are that oxidised MT is converted intonative MT with 2-mercaptoethanol as a reducing agentand Zn2+ as a metal donor, and MT is subsequentlyquantified via Cd saturation. High molecular weight Cd-binding compounds are denatured with acetonitrile, Cubound to MT is removed with ammonium tetrathiomolyb-date and excessive tetrathiomolybdate and its Cu com-plexes are removed with DEAE-Sephacel (Sigma, St.Louis, MO, USA). Subsequently apothionein is saturatedwith 109Cd-labelled CdCl2-solution (Amersham PharmaciaBiotech, Amersham, UK): 50 ppm of 37 MBq mg–1 Cd in0.1 M HCl), and excessive Cd is bound to Chelex 100(Bio-Rad, Munich, Germany). The precipitate wasremoved by centrifugation and the supernatant countedfor 1 min in a gamma counter (Minaxi γ, CanberraPackard, Frankfurt, Germany). The MT concentrationwas calculated assuming a molar ratio of Cd/MT of 7.

Modelling of the data

The modelling of the transfer of contaminants from feed toorgans of growing animals requires detailed informationon organ growth as well as its concomitant Cd accumula-tion. As such information was available for livers andkidneys, a mechanistic transfer model could be developedfor these organs, i.e. a model relating levels in feed toorgan levels due to binding of Cd to MTs. However, in thecase of the blood the absence of growth data of thiscompartment only allowed a strict empirical modellingapproach, i.e. a description of the time course of theaccumulation and depletion kinetics of Cd in the blood.

Food Additives & Contaminants: Part A 3

Red blood cells

Analysis of the plasma samples showed concentrationssystematically below the detection limit of 1 µg kg–1,but analysis of the red blood cell (RBC) samples clearlyshowed an increase of Cd during the exposure phase anddecrease during the phase on clean feed. Therefore, themodelling in blood focussed on the data in RBCs. A one-compartment classical kinetic model for Cd in RBCs wasassumed to be:

CðtÞ ¼ C0 þ ΔCss � ð1� expð�k � tÞÞ t � TC0 þ ΔCss � ð1� expð�k � TÞÞ � expð�k � ðt � TÞÞ T < t

The motivation of such a model is that a continuousbackground contamination during the period precedingthe experimental period has led to a non-zero I initialconcentration of Cd in RBCs at the start of the experi-mental, i.e. C0, and therefore could not be neglected. Afterthe start of the experiment, the concentration increasesduring the exposure period of duration T towards anassumed steady-state elevation level ΔCss due to theuptake of additional Cd ingested through contaminatedfeed. Furthermore, two processes are involved that tendto decrease the concentration with an exponential rate k:dilution due to animal (and thus RBC tissue) growth andelimination due to turnover of RBCs. Due to these pro-cesses the Cd concentration will decrease once the expo-sure to contaminated feed has stopped.

Liver and kidney

In concordance with earlier findings in livestock(Beresford et al. 1999; Crout et al. 2004; EFSA 2004;Van Eijkeren et al. 2007), the accumulation of Cd inorgans like the liver and kidney kinetically may bedescribed as an irreversible uptake process. The transfermodel for Cd in feed to the liver and kidney thereforedescribes the accumulation of Cd in these organs as irre-versible MT binding depending on the dietary amount ofCd taken in (Afeed), or:

dt¼ αAfeed

β þ Afeed�MT (2a)

where dA/dt is the organ-specific Cd uptake rate(mg day–1); MT is the organ-specific metallothioneinconcentration (μmol kg–1); Afeed is the daily Cd intake(mg day–1); β is the parameter characterising saturabletransfer of Cd from feed to MTs (mg day–1); and α isthe parameter scaling the organ MT level to the Cduptake rate (kg µmol–1).(mg day–1). Note that para-meter β has to be interpreted as characterising the over-all saturable binding involved in the transport of Cd

from feed to organ MTs. Next to the MT binding thismay be the (yet unidentified) binding of Cd totransport proteins in the blood, the gastrointestinaltract, etc.

Furthermore note that Equation (2a) as such pre-scribes the continuous accumulation of Cd in the kid-neys and liver as long as the exposure continues. Oncethe exposure is stopped the absolute amount of Cd inthese organs basically remains constant, unless decreas-ing due to organ growth in the period after theexposure.

Integrating Equation (2a) yields the absolute amount(A, mg) of Cd in the liver and the kidney, which may beconverted to the organ Cd concentration (C, mg kg–1 wettissue) by division through organ weight (W), or:

C ¼ A=W (2b)

Being suited for growing pigs the model includes a linearmodel for organ growth, or:

W ¼ W0þ dW : t (2c)

where W0 is the organ weight (kg) at the start of theexposure period; dW is the organ growth rate (kg day–1);and t is the duration of the exposure (days). As shown inthe Supplementary data online (Figure S1), organ weights(W) increase linearly in time during the experimentalperiod.

With regard to MT concentration, Verma et al. (1978)found a linear relationship for organ MT with organ Cdconcentration for a wide range of Cd concentrations inkidney and liver of cow, chicken and swine with correla-tions of 0.90 and 0.43 in swine kidney and liver, respec-tively. So, for the MT concentration a linear model wasassumed, or:

MT ¼ MT0þ dMT :C (2d)

where MT0 is the MT concentration (μmol kg–1 wetweight) at the start of the exposure period and level beforethe treatment; and dMT (μmol kg–1 wet weight day–1) isthe rate at which the MT level changes as function of theorgan Cd concentration during the treatment period(regression parameter).

The carryover rate (COR) is defined as the ratiobetween the organ-specific uptake rate (dAorgan/dt) andthe daily Cd intake (dI/dt), or:

COR ¼ dAorgan=dt

dI=dt(3a)

Equation (2a) can be interpreted in terms of the COR as:

COR ¼ αβ þ Afeed

�MT (3b)

Note that in the present study COR is not constant butdepends on Afeed and MT.

Statistical analysis

Observed differences were tested for significance usingthe Student’s t-test and based on a two-sided comparison.

Results and discussion

Cadmium levels in feed

In principle, five different types of feed were prepared: ablank feed, two feeds with CdCl2 and two with Cd-cysteine (CdCys), aiming for both types of Cd at levelsof 1 and 10 mg Cd kg–1. Regarding the age of the animals,both starter and grower feeds were prepared for each typeof feed. Levels in the starter feed were determined to be0.06 mg kg–1 in the blank feed, 0.78 and 9.5 mg kg–1 inthe feeds with CdCl2, and 1.00 and 9.4 mg kg–1 in thefeeds with CdCys. Levels in the grower feeds were deter-mined to be 0.88 and 10.0 mg kg–1 in the feeds withCdCl2, and 0.84 and 10.0 mg kg–1 in the feeds withCdCys. Levels are expressed on product base but moisturecontent varied between 11.2% and 11.6%, i.e. very closeto the 12% which is the basis for the European Union limitof 0.5 mg kg–1. Therefore, in principle, feed levels werearound the intended levels.

Feed consumption

The animals received the feed ad libitum but the amountconsumed per cage of three animals was determined after3, 8 and 12 weeks and converted to an average feedconsumption per animal (Online Supplementary TableS1). Due to the social housing type, it was not possibleto determine the feed consumption for each individualanimal. Feed consumption in the control animals kept forthe whole 12 weeks increased from 1.27 kg/animal day–1

during the first 3 weeks to 2.03 in weeks 3–8 and 2.70 inthe final 4 weeks.

During the first period of 3 weeks there was no cleartreatment related effect on feed consumption. In this per-iod a larger number of animals received the same treat-ment with average consumptions for the control of1.34 kg/pig day–1 (two cages, six pigs), 1.38 and1.45 kg/pig day–1 for the 1 and 10 mg kg–1 CdCl2 groups(four cages, 12 pigs each treatment) and 1.36 and 1.36 kg/pig day–1 for the 1 and 10 mg kg–1 CdCys groups (fourcages, 12 pigs each treatment). During the second period(3–8 wks) there also seemed to be no consistent differ-ences between the groups. However, in the third period

(8–12 wks), the feed consumption in the two groupsexposed to 10 mg kg–1 for the whole 12-weeks period(coded 12/0) seemed clearly lower than that of either thecontrol pigs, the pigs on the lower dose or the pigs thatswitched to clean feed after 3 weeks. However, there wasquite some variation in the data and it is difficult to drawfirm conclusions.

Based on feed levels and intake, the total intake of Cdper pig during the 3, 8 or 12 weeks of the study werecalculated (Online Supplementary Table S1), with thelargest intake for the two groups fed on 10 mg kg–1

CdCl2 or CdCys for 12 weeks, both being around 1.5gper pig.

Body and organ weights

Online Supplementary Table S2 shows the body weight ofthe pigs at the time of slaughter, as well as the absoluteand relative weights of livers and kidneys. Control pigsgrew from about 20 kg before the start of the treatment to38 and 97 kg after 3 and 12 weeks respectively. Pigs werealso regularly weighed during the study, allowing moni-toring of the growth of individual animals for the wholestudy period, i.e. those not being slaughtered at intermedi-ate times. Figure 2 shows the progression of the bodyweights of the untreated pigs and the pigs exposed to the

0 5 10 15

Time (week)

control1 mg/kg (12 wks)1 mg/kg (3 + 9 wks)10 mg/kg (12 wks)10 mg/kg (3 + 9 wks)

Figure 2. Body weights of control pigs, pigs exposed to 1 or10 mg kg–1 CdCl2 or Cd-cysteine for 12 weeks, or pigs exposedfor 3 weeks to 1 or 10 mg kg–1 followed by 9 weeks on cleanfeed. Since the body weights were similar for CdCl2 or Cd-cysteine, data were combined (mean ± SD for n = 3 (controls)or n = 6 (exposed) except for the 10 mg kg–1 group for 12 weekswhere n = 5 due to 1 animal dropping out). aSignificantlydifferent from that of the combined control and low dosed pigs(p < 0.05, Student’s t-test, two-sided).

high and low dose of CdCl2 or CdCys for 12 weeks, or3 weeks followed by a 9-week period on clean feed.Regarding the absence of clear differences in body weight,the data for CdCl2 or CdCys were combined. The pigsexposed to the dose of 10 mg kg–1 feed for 12 weeks,showed less growth during the last 5 weeks. This differ-ence was not significant (p = 0.17) when compared withthe three control pigs but highly significant (p = 0.00004)when compared with the combined controls and low-dose(1 mg kg–1) groups. This was not the case for the pigsfrom the high-dose group that switched after 3 weeks toclean feed (p = 0.66).

In the case of 10 mg kg–1 CdCl2, weight increase inthis last period was 19.9 ± 3.4 kg, as compared with29.5 ± 2.2 kg in the controls and 29.5 ± 2.2 and27.3 ± 3.1 kg in the animals exposed to 1 mg kg–1 feedfor 3 (+9 weeks clean feed) or 12 weeks respectively. Theanimals exposed to the feed with 10 mg kg–1 for 3 weeksfollowed by 9 weeks on clean feed, showed an increase of22.9 ± 2.5 kg, again less than the control and low dose(1 mg kg–1) groups. In the case of CdCys, the pigsexposed to the high dose also showed a decreased growthduring the last 3 weeks, being 18.2 ± 2.8 kg, but in thiscase the group exposed for only 3 weeks followed by9 weeks on clean feed showed a normal growth of26.0 ± 9.1 kg and the animals exposed to 1 mg kg–1 for3 and 12 weeks showed a growth of 24.4 ± 1.7 and28.7 ± 1.2 kg respectively. This decreased growth wasaccompanied by a reduced feed intake but also anincreased feed conversion ratio (Online SupplementaryTable S1). An increased feed conversion ratio was alsoobserved in the last period for the group exposed to10 mg kg–1 CdCl2 for 3 weeks followed by clean feedfor 9 weeks, being the group that also showed a decreasedgrowth.

The liver and kidney weights reflected the bodyweights, showing a reduction in the case of the animalsexposed to the high dose of CdCl2 or CdCys for the wholeperiod (Online Supplementary Table S2).

Overall these data indicate that prolonged feeding withfeed containing 10 mg kg–1 Cd causes some kind ofadversely changed growth physiology in the animals,leading to a reduced growth. Alternatively, pigs are moresusceptible to Cd during this last period of 4 weeks.Phillips et al. (2003) treated pigs for 137 days and alsoobserved a decreased growth and increased feed conver-sion ratio at a dose of 2.5 mg kg–1 feed. Similar was truefor Xu et al. (2004) who exposed pigs for 12 weeks to10 mg kg–1 Cd in the feed for 12 weeks and observed aslight decrease in feed intake but a clearly decreasedgrowth and increased feed conversion ratio. However,King et al. (1992) fed pigs a wheat-based feed contami-nated with CdCl2 for their whole lifetime (8–90 kg) withCd levels up to 4.4 mg kg–1 but did not observe any effecton feed intake or growth rate. Tu et al. (2007) treated pigs

for 12 weeks and observed an increased relative liverweight at a dose level of 10 mg kg–1, as well as anincrease in serum glutamic-pyruvic transaminase (GPT)and decreased Na+/K+-ATPase activity in liver, both indi-cative of liver damage. Unfortunately, body weights andfeed consumption were not reported. In the current studyno effect on relative liver weight was observed (OnlineSupplementary Table S2). Nevertheless, these studies indi-cate that the higher dose levels may to some extent affectpigs and that the current European Union feed limit of0.5 mg kg–1 also protects pigs for these potential adverseeffects of Cd.

For the transfer model (Equation 2) for the Cd con-centration in liver and kidney, an organ weight model wasrequired. Online Supplementary Figure S1 shows the liverand kidney weights at 3, 8 and 12 weeks after the start ofthe experiment. When combining all animals, the linearmodels describing the data are: liver weight(g) = 626 + 100 × time (weeks); kidney weight(g) = 114 + 26 × time (weeks).

Metallothioneins in kidneys and liver

Exposure to heavy metals is known to trigger the forma-tion of MTs which subsequently bind the metals. Sincethis phenomenon is important for modelling the data, MTlevels were determined in livers and kidneys of all ani-mals. Again, there were no clear differences between theCdCl2 or CdCys groups and therefore data were com-bined. As shown in Figure 3 (upper left), MT levels inkidneys of control animals decreased with age from aninitial 12.5 ± 0.4 µmol kg–1 at the start of the experimentto 10.0 ± 3.4 µmol kg–1 and 6.2 ± 1.0 µmol kg–1 after anadditional 3 and 12 weeks. Exposure to the high Cd dose(10 mg kg–1) resulted in increased levels of 14.8 ± 2.1 and20.4 ± 0.5 µmol kg–1 after 3 and 12 weeks. When theexposure was stopped after 3 weeks, levels decreased to13.0 ± 1.7 and 10.5 ± 1.2 µmol kg–1 at 8 and 12 weeks,the latter still being almost double the level in the controlgroup. This is in line with the increased Cd levels in thesekidneys (see below). In the animals exposed for 12 weeksto the low dose (1 mg kg–1), the MT levels at 3 and12 weeks were respectively 10.3 ± 1.7 and9.2 ± 1.4 µmol kg–1, meaning no difference at 3 weeksbut not showing the further decrease observed in thecontrols at 12 weeks. When exposed to this low dose foronly 3 weeks, followed by clean feed, there was nodifference with the control animals at 12 weeks.

In the case of the livers (Figure 3, upper right), initialMT levels at the start of the study were five-fold higherthan in kidneys, being 60.0 ± 7.4 µmol kg–1. Levels weresimilar to those reported for pig livers by Henry et al.(1994). MT levels decreased to 22.9 ± 3.0 and19.5 ± 3.8 µmol kg–1 after 3 and 12 weeks in the controlanimals. In the animals exposed to the highest Cd levels

for 3 and 12 weeks, levels were respectively 23.0 ± 3.6and 23.4 ± 4.3 for CdCl2, and 16.5 ± 2.5 and20.7 ± 3.8 µmol kg–1 for CdCys. So contrary to thekidneys, there was no clear effect of the high Cd exposureon the MT levels in livers. López-Alonso et al. (2012)investigated Cd and MT levels in pigs raised under inten-sive and extensive production systems. MT levels in liverswere similar between the two groups, but in kidneys theMT levels were 50% higher in the intensively reared pigs,which was attributed to the difference in supplementationwith zinc and copper. Cd levels in both livers and kidneyswere much higher in intensively reared pigs. In general,MT levels in livers were much higher than in kidneys.

In order to model the Cd levels in liver and kidney, amodel for Cd induced MT levels in these organs isrequired (Equation 2). Figure 3 (lower panels) showsthe relation between Cd and MT levels in kidneys (left)

and livers (right). In kidneys, regression was applied ofMT levels in dependence of Cd levels following thelinear MT induction model of Equation (2), resulting invalues for MT0 = 8.65 (SD = 0.37) and dMT = 0.923(SD = 0.081). Based on this model, for the lowest doseapplied, the MT levels were estimated to be 9.1 µmol kg–1

after 3 weeks and 9.0 and 10.0 µmol kg–1 after 12 weeksfor halted (3 + 9) and continued exposure (12 + 0),respectively. For the highest dose applied, MT levelswere 13.1 µmol kg–1 after 3 weeks and 10.6 and21.0 µmol kg–1 after 12 weeks for halted and continuedcontamination, respectively. In liver, the regression coef-ficient –0.9 (SD = 1.8) for the estimated MT levels independence of Cd levels appeared to be insignificant.Mean MT level in the liver is 22.3 µmol kg–1 with astandard deviation of 12.2, reflecting the large variabilityin MT levels detected.

Time (week)

control1 mg/kg (12 wks)1 mg/kg (3 + 9 wks)10 mg/kg (12 wks)10 mg/kg (3 + 9 wks)

Cd concentration (mg/kg)

0 5 10 15

Time (week)

0 5 10 15

0 1 2 3 4

Cd concentration (mg/kg)

Figure 3. Levels of metallothioneins in kidneys (upper left) and livers (upper right) of (1) control pigs, or pigs exposed to CdCl2 or Cd-cysteine at (2) 1 mg kg–1 for 12 weeks, (3) 1 mg kg–1 for 3 weeks followed by 5 or 9 weeks on clean feed, (4) 10 mg kg–1 for 12 weeksor (5) 10 mg kg–1 for 3 weeks followed by 5 or 9 weeks on clean feed (3 + 9). Since the MT concentrations were similar for CdCl2 andCd-cysteine, data were combined (mean ± SD for n = 3 (controls) or n = 6 (exposed)). a,bStatistically significant different from control (a)or continued (b) exposure (p < 0.05, two-sided Student’s t-test). Lower panels show the relation between concentrations of Cd and MT inkidneys (left panel, linear relation) and livers (right panel, constant).

Cadmium levels in liver and kidneys

Table 1 shows the levels of Cd in the livers and kidneys ofthe animals, both as concentration and as absoluteamounts based on the organ weights; OnlineSupplementary Figure S2 shows the combined data forpigs treated with CdCl2 and CdCys. It is evident that atboth doses in the feed there is a time-related increase inthe levels in both tissues with on average a four- to five-fold higher level in the kidneys. Such differences werealso reported by other groups and shows that kidney is themajor organ in terms of concentrations (Bache et al. 1987;King et al. 1992; Zacharias et al. 2001) (OnlineSupplementary Table S5). The levels after 12 weeks onthe contaminated feeds were roughly three times higherthan at 3 weeks. The 10-fold difference in the levels of thelow and high dose is also reflected in the levels in bothkidneys and livers at 3 and 12 weeks, possibly with theexception of the high CdCys group at 12 weeks. Levelsobserved by King et al. (1992) in animals fed a CdCl2fortified diet were similar for livers but up to two timeshigher in kidneys. Zacharias et al. (2001), also supplyingCdCl2 but slaughtering the animals at 30 and 50 kg,observed much higher levels in both liver and kidneys.These authors demonstrated that other components in thefeed like phytase and calcium might have an influence onthe bioaccessibility and hence absorption of Cd in thegastrointestinal tract. At the same time, in the present

study, kidney levels in the control animals after12 weeks (0.08 mg kg–1) were much lower than observedby previous authors, being 0.78 (Bache et al. 1987), 0.39(King et al. 1992), and 0.6 mg kg–1 (Zacharias et al. 2001)despite the similar levels in the control feed, being around0.06 mg kg–1. This suggests that other sources than thefeed could also have contributed to the exposure in someof these studies or that the animals already containedhigher levels at the start of the study.

During the exposure period, the weights of livers andkidneys increased both by a factor of 2–2.5 between 3 and12 weeks. Table 1 also shows the total amount of Cd inthe livers and kidneys, demonstrating that livers and kid-neys contain similar absolute amounts. Between weeks 3and 12, there is a six- to eight-fold increase in the absoluteamounts in the livers and kidneys in the low dosagegroups, but in the high dosage groups this relative increaseis less, being five- to six-fold. This may be related to thedifferences in the lower increase in liver and kidneyweights in the higher dosage groups. When comparedwith the total intake of Cd, the relative amounts retainedin liver and kidneys in the low-dose groups were respec-tively 0.47% and 0.44%, and in the high-dose groups0.36% and 0.34%.

Following the switch at 3 weeks to clean feed, therewas initially an increase in the absolute amounts in bothkidneys and livers in some of the groups, like the low dose

Table 1. Concentrations and absolute amounts of Cd in livers and kidneys of pigs (mean ± SD). The European Union limits are 0.5 and1 mg kg–1 respectively.

Period

(weeks) Liver Kidney

Dose (mg kg–1 feed) Cd Clean Level (mg kg–1) Total (mg) Level (mg kg–1) Total (mg)

Control 0 0 0.03 ± 0.01 0.013 ± 0.002 0.13 ± 0.05 0.009 ± 0.0060 3 0.01 ± 0.01 0.013 ± 0.005 0.09 ± 0.01 0.016 ± 0.0090 12 0.02 ± 0.01 0.030 ± 0.016 0.08 ± 0.03 0.033 ± 0.009

CdCl2, 1 mg kg–1 3 0 0.10 ± 0.03 0.087 ± 0.024 0.44 ± 0.10 0.080 ± 0.01912 0 0.34 ± 0.07 0.660 ± 0.158 1.53 ± 0.21 0.651 ± 0.0623 5 0.13 ± 0.02 0.175 ± 0.035 0.51 ± 0.12 0.158 ± 0.0343 9 0.08 ± 0.03 0.150 ± 0.031 0.38 ± 0.13 0.153 ± 0.048

CdCl2, 10 mg kg–1 3 0 1.03 ± 0.15 1.025 ± 0.116 5.00 ± 0.79 1.033 ± 0.23312 0 3.20 ± 0.26 5.620 ± 0.384 14.67 ± 1.15 5.444 ± 0.2943 5 0.76 ± 0.14 1.094 ± 0.149 3.53 ± 0.25 1.176 ± 0.1953 9 0.50 ± 0.09 0.855 ± 0.209 1.80 ± 0.14 0.808 ± 0.184

CdCys, 1 mg kg–1 3 0 0.12 ± 0.02 0.113 ± 0.009 0.46 ± 0.07 0.082 ± 0.00912 0 0.37 ± 0.05 0.724 ± 0.099 1.43 ± 0.15 0.652 ± 0.0433 5 0.09 ± 0.02 0.132 ± 0.024 0.39 ± 0.05 0.136 ± 0.0213 9 0.09 ± 0.02 0.158 ± 0.049 0.36 ± 0.15 0.148 ± 0.092

CdCys, 10 mg kg–1 3 0 0.94 ± 0.04 0.870 ± 0.073 4.60 ± 0.78 0.892 ± 0.19912 0 3.35 ± 0.07 5.409 ± 0.037 11.50 ± 0.71 4.259 ± 0.1683 5 0.76 ± 0.21 1.045 ± 0.344 3.23 ± 0.60 1.014 ± 0.2283 9 0.64 ± 0.19 1.182 ± 0.035 2.30 ± 0.36 1.013 ± 0.107

CdCl2 group (Table 1). There was hardly any change inthe next 4 weeks, neither in kidneys nor in livers. Thesuggestion that Cd binds to MT in the liver and is thentransported to the kidneys, where it accumulates seemsthus very unlikely.

The model in Equation (2), given the organ growthmodels and the MT induction models described above,was applied for fitting the parameters α and β to thedata on the amounts after 3 and 12 weeks continuousexposure for both liver and kidney. The fitted values forthe model parameters are α = 7.2 × 10−3 (SD =0.7 × 10−3) and β = 12.4 (SD = 2.0) for kidney andα = 8.4 × 10−3 (SD = 1.3 × 10−3) and β = 237.0 (SD =7.4) for liver. Data on the absolute amounts of Cd inkidney and liver for the animals exposed for a contin-uous period of 3 or 12 weeks are compared in OnlineSupplementary Figure S3 with the resulting model.Modelled data for the resulting concentrations in kid-neys and livers at the two doses of CdCl2 and CdCysare depicted in Figure 4, showing a good fit both duringcontinuous exposure and after the switch to clean feed.

The latter data may serve as a model verification. Thesegraphs take into account the growth-related increase inthe organ weights.

Studies in different animal species have shown a lowabsorption of ingested Cd (0.5–7%) (EFSA 2004). Theresults of the present study are in line with such lowabsorption and, hence, a low carryover to the animal.For example, given the model parameterisation (Equation2a) the daily uptake in the kidney amounts up to0.1 mg day–1 in animals exposed for 12 week to feedcontaining 10 mg Cd kg–1. Given a daily intake of22 mg Cd this corresponds with a COR of 0.45%.

The calibrated model was used to simulate the accu-mulation of Cd in the kidneys and the liver for feedcontaining 0.5 mg kg–1. The result of this simulation,which is shown in Figure 5, indicates that Cd levels inkidneys of pigs exposed to Cd at the European Unionlimit in feed will exceed the limit after 12 weeks ofexposure. In the case of the liver the European Unionlimit in feed prevents that, even after a prolonged expo-sure period.

0 5 10 15Time (week)

Time (week)

0 5 10 15

Time (week)

Figure 4. Comparison of the model for the concentration of Cd (line) and experimental data (closed circles) in kidney (left panels) andliver (right panels). Upper panels: 1 mg kg–1 feed contamination level; lower panels: 10 mg kg–1 feed contamination level.

Cadmium levels in other tissues and hair

Levels in muscle tissue in all dosage groups were belowthe LOQ of 0.01 mg kg–1, which is five times under themaximum limit for Cd in the European Union (data notshown). This is in line with previous studies (Lisk et al.1982; Bache et al. 1987; Sapunar-Postružnik et al. 2001;Zacharias et al. 2001). Similar was true for the brains,showing no detectable Cd levels in any of the groups.

There was a slight increase in the Cd levels in thetestes of the high dosage groups after 3 and 12 weeks,being 0.020 ± 0.010 and 0.049 ± 0.007 mg kg–1 for theCdCl2 group, and 0.023 ± 0.003 and 0.046 ± 0.026 mg kg–1

for the CdCys group as compared with non-detectable(<0.010) in the control group (Online SupplementaryTable S1). The lungs in these dosage groups showed Cdlevels of 0.083 ± 0.032 and 0.079 ± 0.002 at week 3 and0.230 ± 0.040 and 0.167 ± 0.084 mg kg–1 at week 12.Levels decreased upon the switch to clean feed at 3 weeksbut were still detectable after 9 weeks (OnlineSupplementary Table S1). Phillips et al. (2003) also exam-ined Cd levels in testes and lungs of pigs exposed to dosesup to 2.5 mg Cd kg–1 feed for 137 days, but observed levelsbelow 0.003 mg kg–1 in these tissues, despite some dose-related increase.

As shown in Online Supplementary Table S2, levelsin the duodenum were clearly increased in the high-dosage groups at 3 and 12 weeks, 4.30 ± 2.80 and1.12 ± 0.17 mg kg–1 for the CdCl2 group, and2.36 ± 2.38 and 3.10 ± 1.31 mg kg–1 for the CdCysgroup as compared with 0.04 ± 0.02 and 0.05 ± 0.02 inthe control group. Also in the low-dose groups, levelswere increased, being 0.61 ± 0.09 and 0.98 ± 0.13 mg kg–1

for the CdCl2 group, and 0.62 ± 0.17 and0.37 ± 0.18 mg kg–1 for the CdCys group. In the high-

dose groups this was still the case after 5 or 9 weeks onclean feed, indicating that the increase is not only due topossibly remaining feed in the samples. Also in the ileumand jejunum levels in the high-dose groups were clearlyincreased compared with the controls (OnlineSupplementary Table S2).

Hair samples were also collected at the time of slaugh-ter but levels were very variable and it was difficult todraw conclusions (Online Supplementary Table S1), espe-cially due to a high level in one pig in the control group.When focusing on the pigs slaughtered after 12 weeks,levels in hair of the exposed animals are somewhat ele-vated, but there is no clear dose-related increase. Overall,this questions the use of hair as an indicator of exposure,as was suggested by data from other studies (King et al.1992; Phillips et al. 2003), although these studies alsoshowed variable results.

Cadmium levels in blood

Concentration data of Cd in RBCs for the high-dosegroups are shown in Figure 6. For each of the two Cdforms, CdCl2 and CdCys, the data showed similar results.All data together of the pigs exposed during 3 weeks to10 mg Cd kg–1 feed were taken to fit the model parametersof the model Equations (1) and (2). The model parametersthus obtained are the background concentration C0 = 0.11(SD = 0.08) µg kg–1, the steady-state elevation levelΔCss = 14.0 (SD = 0.3) µg kg–1 and the dilution/elimina-tion rate k = 0.0272 (SD = 0.0008) day–1. The model wasalso verified on the data obtained for the animals with aprolonged exposure of 12 weeks. Figure 6 (left panel)shows a clear fit for the data obtained for the high-dosegroups exposed for 12 weeks, reaching a plateau after

Time (week)

0 5 10 15

Time (week)

Figure 5. Model simulation of the time-course of the Cd concentration in the kidneys (left panel) and the liver (right panel) after feedingcontaminated feed at a level of 0.5 mg kg–1 (European Union limit). Horizontal lines: European Union limits for Cd in the kidneys(1.0 mg kg–1 wet weight) and liver (0.5 mg kg–1 wet weight).

about 15 weeks. When stopping the exposure after3 weeks, the levels decreased gradually and reached back-ground levels after about 15 weeks. The model was alsoverified on the data obtained from the continued Cdadministration experiment of 1 mg kg–1 feed (Figure 6,right panel). Verification was obtained by multiplication ofΔCss by the ratio 0.0875 of the total applied Cd of the low-and high-dose regimen. Although most of the levels werearound the LOQ for RBCs, the verifications show a faircomparison between the model and the data.

Conclusions

The present study shows the time- and dose-relatedincrease in Cd levels in various tissues of growing pigs,in most cases in line with previous studies. The use ofdifferent exposure periods and the switch to clean feedallowed the modelling of the data, leading to a mechan-istic model that can be used in future incidents with Cdbut also helps to understand the behaviour of Cd in grow-ing pigs. Following the switch to clean feed, the decreasein the levels in liver and kidneys could entirely beexplained by the further growth of the animals.

There was no clear difference between the twoapplied forms of Cd, suggesting that at least theseforms show similar absorption, distribution, metabolismand excretion (ADME) properties in pigs. Other authorsfed pigs with plant materials grown on contaminatedfields and observed levels in livers and kidneys thatwere in a similar range as observed with CdCl2.Therefore, these studies do not indicate a difference inthe bioavailability of different forms of Cd, assuming

that most of the Cd was present in a bound form in theplant materials.

In domestic animals the largest fraction of Cd isfound in the liver and kidneys. The present study con-firms this finding, with Cd concentrations in the liver andkidneys exceeding those in the rest of the body manifold.However, preferential Cd accumulation in the liver overthe kidneys with increasing exposure (EFSA 2004) wasnot found in the present study. On the contrary, theaccumulation of Cd in the liver and kidneys showedsimilar dynamics (Online Supplementary Figures S3 andS4), however with the concentration in the kidneysexceeding that in the liver by a factor of four. The lattereffect is in concordance with the MT findings in theseorgans. Despite organ growth there was an increase ofthe MT level in the kidneys but a decrease in the MTlevel in the liver at increasing exposure (Figure 3). Thesefindings indicate an independent role of the kidneys inCd accumulation, i.e. kidney-specific MT synthesis fol-lowed by Cd binding to the induced MT proteins.Alternatively, it has been suggested that the exposure toCd may lead to the binding of Cd2+ to serum albuminwhich may be converted to the Cd–MT complex in theliver. Circulating Cd–MT complexes then may reach thekidneys, where they may be filtered by the glomerulusand reabsorbed by the proximal tubule cells (EFSA2004). Though the results of the present study certainlydo not exclude the latter disposition mechanism, as said,they also hint on the existence of a kidney-specific Cdbinding mechanism, i.e. the induction of kidney-specificMT proteins followed by the binding of Cd in the bloodto the induced proteins.

Time (week)

0 5 10 15Time (week)

0 5 10 15

Figure 6. Cd concentrations in red blood cells (RBC) of six animals, three on CdCl2 and three on CdCys, both at about 10 mg Cd perkg feed. The model was fit to the data from animals exposed during 3 weeks only (lower line, closed triangles). Exposure of the otheranimals was ended after 12 weeks resulting in increased levels that served the model verification (upper line, closed circles). The LOQwas determined as 1 ng Cd g–1 RBC. Nevertheless, indicative values for lower exposure levels were reported and plotted too (rightpanel).

Overall, the current feed limit in the European Unionof 0.5 mg kg–1 prevents that level in kidneys and liversexceeding the limits, provided that the exposure period islimited to fewer than 12 weeks.

AcknowledgementThe authors would also like to acknowledge the work of theanimal care takers at ASG.

FundingThis study was financed by the Ministry of Economic Affairs(EZ), the Ministry of Health, Welfare and Sport (VWS), and theNetherlands Food and Consumer Products Safety Authority(NVWA).

Supplemental dataSupplemental data for this article can be accessed here: http://dx.doi.org/10.1080/19440049.2014.979370.

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Carryover of cadmium from feed in growing pigs

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