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Physiologically Based Pharmacokinetics of ZearalenoneBeom Soo Shin a , Seok Hyun Hong b , Jürgen B. Bulitta c , Jong Bong Lee b , Sang WookHwang b , Hyoung Jun Kim b , Seung Du Yang a , Hae-Seong Yoon d , Do Jung Kim d , Byung MuLee b & Sun Dong Yoo ba College of Pharmacy, Catholic University of Daegu, Gyeongsan-si, Gyeongbuk, Koreab College of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do, Koreac Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences,University at Buffalo, State University of New York, Buffalo, USAd Human Exposure Assessment Division, National Institute of Toxicological Research,Eunpyung-ku, Seoul, KoreaPublished online: 29 Dec 2009.

To cite this article: Beom Soo Shin , Seok Hyun Hong , Jürgen B. Bulitta , Jong Bong Lee , Sang Wook Hwang , HyoungJun Kim , Seung Du Yang , Hae-Seong Yoon , Do Jung Kim , Byung Mu Lee & Sun Dong Yoo (2009) Physiologically BasedPharmacokinetics of Zearalenone, Journal of Toxicology and Environmental Health, Part A: Current Issues, 72:21-22,1395-1405, DOI: 10.1080/15287390903212741

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Journal of Toxicology and Environmental Health, Part A, 72: 1395–1405, 2009Copyright © Taylor & Francis Group, LLCISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287390903212741

1395

UTEHPhysiologically Based Pharmacokinetics of ZearalenonePbpk Modeling of ZearalenoneBeom Soo Shin1, Seok Hyun Hong2, Jürgen B. Bulitta3, Jong Bong Lee2,

Sang Wook Hwang2, Hyoung Jun Kim2, Seung Du Yang1, Hae-Seong Yoon4, Do Jung Kim4, Byung Mu Lee2, and Sun Dong Yoo2*

1College of Pharmacy, Catholic University of Daegu, Gyeongsan-si, Gyeongbuk, Korea, 2College of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do, Korea, 3Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, USA, and 4Human Exposure Assessment Division, National Institute of Toxicological Research, Eunpyung-ku, Seoul, Korea

The objectives of this study were to (1) develop physiologicallybased pharmacokinetic (PBPK) models for zearalenone followingintravenous (iv) and oral (po) dosing in rats and (2) predictconcentrations in humans via interspecies scaling. The model foriv dosing consisted of vein, artery, lung, liver, spleen, kidneys,heart, testes, brain, muscle, adipose tissue, stomach, and smallintestine. To describe the secondary peak phenomenon observedafter po administration, the absorption model was constructed toreflect glucuronidation, biliary excretion, enterohepatic recircula-tion, and fast and slow absorption processes from the lumenalcompartment. The developed models adequately describedobserved concentration–time data in rats after iv or po adminis-tration. Upon model validation in rats, steady-state zearalenoneconcentrations in blood and tissues were simulated for rats afteronce daily po exposures (0.1 mg/kg/d). The average steady-stateblood zearalenone concentration predicted in rat was 0.014 ng/ml.Subsequently, a daily human po dose needed to achieve the samesteady-state blood concentration found in rats (0.014 ng/ml) wasdetermined to be 0.0312 mg/kg/d or 2.18 mg/70 kg/d. The steady-state zearalenone concentration–time profiles in blood and tissueswere also simulated for human after multiple po administrations(dose 0.0312 mg/kg/d). The developed PBPK models adequatelydescribed the pharmacokinetics in rats and may be useful inpredicting human blood and tissue concentrations for zearalenoneunder different po exposure conditions.

Zearalenone is a mycotoxin biosynthesized by fungi belongingto the genus Fusarium, including F. graminearum, F. culmorum,F. equiseti, and F. cerealis (Kuiper-Goodman et al., 1987;

Bennett & Klich, 2003). These species are ubiquitously foundas contaminants in grains and animal feeds; consequently, ani-mals and humans are at risk of being exposed to zearalenone(Kuiper-Goodman et al., 1987; Jodlbauer et al., 2000; Kleinovaet al., 2002; Zöllinger et al., 2002; Songsermsakul et al., 2006;Zinedine et al., 2007). Zearalenone exhibits a relatively lowacute toxicity (oral LD50 2000–20,000 mg/kg in mice, rats, andguinea pigs) (Baldwin et al., 1983; Zinedine et al., 2007). How-ever, zearalenone binds to estrogen receptors (ER) (Boyd &Wittliff, 1978; Everett et al., 1987) and may produce variousestrogen-disrupting effects, such as infertility, reduced serumtestosterone concentrations and sperm counts, enlargement ofovaries and uterus, reduced incidence of pregnancy, and alter-ations in progesterone concentrations in animals, at relativelylow levels (Bennett & Klich, 2003; Döll et al., 2003; Zinedineet al., 2007). Thus, in the year 1996, 6 countries regulatedzearalenone, and it was regulated by 16 countries by 2003(FAO, 2004). The maximum allowable amount of zearalenonein foods and feeds ranges from 50 to 1000 μg/kg in variouscountries (FAO, 2004), and a total daily intake of 0.1 μg/kgwas proposed as a margin of safety (MOS) in humans (Kuiper-Goodman et al., 1987). The pharmacokinetics of zearalenonewas reported in animals, including pigs (Prelusky et al., 1989;Biehl et al., 1993; Dänicke et al., 2005), broilers (Dänickeet al., 2001), and rats (Mallis et al., 2003; Shin et al., 2009, thisissue). Limited information is available on the pharmacokineticsof zearalenone in man (Mirocha et al., 1981).

As human pharmacokinetic data on environmentally toxicchemicals are scarce, physiologically based pharmacokinetic(PBPK) modeling is a rational and ethical alternative approachto extrapolate human pharmacokinetics. Unlike conventionalpharmacokinetic models, PBPK models more specificallydescribe the pharmacokinetics and tissue distribution by incor-porating actual volumes of physiological fluids and tissues,blood flow rates, and tissue-to-blood partition coefficients.

This work was supported by the Korea Food and DrugAdministration, grant 08182–488.

Address correspondence to Sun Dong Yoo, PhD, Professor,College of Pharmacy, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon, Gyeonggi-do, 440-746, Korea. E-mail:sdyoo@skku.ac.kr

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1396 B. S. SHIN ET AL.

These models are useful to evaluate pharmacokinetic changesas a result of alterations in certain pathophysiologic conditions.Perhaps mostly importantly, PBPK models may be applied topredict the pharmacokinetics and tissue distribution fromseveral animal species to human (Shin et al., 2004). In thecourse of risk assessment of zearalenone in our laboratory, thedisposition and dose-linearity of pharmacokinetics, tissue dis-tribution, oral bioavailability, and urinary and biliary excretionof zearalenone were studied in rats (Shin et al., 2009, thisissue). The objectives of the present study were to (1) developand qualify PBPK models for zearalenone in rats after intrave-nous ( iv) and oral (po) administration and (2) predict zearale-none concentrations in humans in blood and various tissuesafter multiple po intake.

MATERIALS AND METHODS

ChemicalsZearalenone, zearalanone (internal standard), and xylazine

were purchased from Sigma Chemical (St. Louis, MO). Dime-thyl sulfoxide (DMSO) and polyethylene glycol (PEG) 400were purchased from Yakuri Pure Chemicals (Kyoto, Japan).Heparin sodium and saline were obtained from Choong WaePharma (Seoul, Korea). Acetonitrile, methanol, t-butyl methylether, and distilled water (all high-performance liquid chroma-tography [HPLC] grade) were purchased from MallinkrodtBaker (Phillipsburg, NJ).

AnimalsMale Sprague-Dawley rats (8–10 wk old, weighing 230–290 g)

were purchased from Hyundai Bio (Ansung, Korea) and keptin plastic cages with free access to water and standard rat diet(Samyang, Seoul, Korea). The rats were maintained in an ani-mal facility at a temperature of 23 ± 2°C with a 12/12-h light/dark cycle and relative humidity of 50 ± 10% for at least 1 wkprior to experimentation. The rats were anesthetized by ipinjection of ketamine and xylazine (90/10 mg/kg) and cannu-lated with PE tubing (0.58 mm ID and 0.96 mm OD, NatumeCo., Tokyo, Japan) in the right jugular and femoral veins for ivinjection and infusion and in the right jugular vein for poadministration studies. For bile fluid collection, rats were addi-tionally cannulated in the bile duct with PE tubing (0.28 mmID and 0.61 mm OD, Natume Co., Tokyo). After surgery, atleast a 1-d recovery was allowed prior to administration ofzearalenone.

Intravenous Bolus and Oral Administration StudyZearalenone dissolved in a mixture of DMSO:PEG

400:saline (1:5:4, v/v) was given by iv injection (dose 8 mg/kg)to intact and bile duct-cannulated (BDC) rats (n = 4 each).Zearalenone was additionally administered to intact and BDCrats (n = 6 and 3, respectively) by po gavage (dose 8 mg/kg).

After iv injection, serial blood samples (approximately 0.3 mleach) were taken via the jugular vein catheter at 0, 5, 10, 15,and 30 min, and 1, 2, 3, 6, 9, and 12 h After po administration,additional blood samples were taken at 1.5 and 24 h after dosing.Serum samples were harvested by centrifugation at 3500 × g(Micro V, Fisher Scientific Co., Pittsburgh, PA) for 10 min. Tocalculate the blood-to-serum partition coefficient in rats, wholeblood samples were taken separately after iv injection at eachsampling time. The blood-to-serum partition coefficient wascalculated as whole-blood zearalenone concentration dividedby serum zearalenone concentration. The harvested whole-blood and serum samples were immediately frozen and storedat −20ºC until analysis. Bile samples were collected from BDCrats over 0–30 and 30–60 min, and 1–1.5, 1.5–2, 2–3, 3–4, 4–6,6–12, and 12–24 h intervals after iv injection. Urine wascollected from rats in metabolic cages over a 12-h periodafter iv injection. Collected bile and urine samples werestored at −20ºC until analysis.

Intravenous Infusion StudyZearalenone dissolved in a mixture of DMSO:PEG

400:saline (1:5:4, v/v) was iv infused to rats (model 200 Infu-sion Pump, KD Scientific, Boston) at 2 different rates of 1.13and 2.25 mg/h/kg (n = 4 and 5, respectively) over 6 h. Theselow and high infusion rates were calculated as the product ofthe systemic clearance of zearalenone determined after iv bolusinjection and the target steady-state serum concentrations of225 and 450 ng/ml, respectively. Blood samples were taken at2, 4, and 6 h during infusion and centrifuged at 3500 × g for10 min. Harvested serum samples were stored at −20°C untilanalysis. At the end of the iv infusion, rats were sacrificed bycervical dislocation, and lung, liver, spleen, kidneys, heart,testes, brain, muscle, adipose tissue, stomach, and small intes-tine were excised, blotted dry, and immediately homogenizedin phosphate buffer (pH 7.4) (PowerGen 125, Fisher ScientificCo., Pittsburgh, PA). The homogenized tissue samples wereimmediately frozen and kept at −20°C for drug analysis.

Zearalenone concentrations in biological samples weredetermined by validated liquid chromatography–mass spec-troscopy (LC/MS/MS) and HPLC/fluorescence assay methodsreported previously. The LC/MS/MS assay was used to deter-mine zearalenone concentrations in serum, bile and urine (Shinet al., 2009a, 2009b), and the HPLC with fluorescence detec-tion (FLD) assay was used to determine zearalenone concen-trations in various tissues (Shin et al., 2009a, 2009b).

Development of PBPK ModelsSchematic representations of the PBPK models used for iv

injection and po administration of zearalenone are shown inFigure 1. These models consisted of vein, artery, lung, liver,spleen, kidneys, heart, testes, brain, muscle, adipose tissue,stomach, and small intestine. The oral absorption model wasconstructed to reflect glucuronidation, biliary excretion,

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PBPK MODELING OF ZEARALENONE 1397

enterohepatic recirculation, and fast and slow absorption pro-cesses from lumen. The lumen compartment was added to themodel to describe the biliary recirculation of zearalenone viafast and slow absorption processes. Differential mass balanceequations were written for individual tissues as below. For ivinjection, Eq. (1) shows the mass balance for venous blood,Eq. (2) for arterial blood, Eq. (3) for noneliminating tissues(spleen, kidneys, heart, testes, brain, muscle, adipose tissue,stomach, and small intestine, Eq. (4) for eliminating tissue(liver), and Eq. (5) for lung.

• For venous blood:

• For arterial blood:

• For noneliminating tissues:

• For an eliminating tissue (liver; with QHepatic artery =QLi - QSp - QSt - QSI):

FIG. 1. Schematic diagrams of the PBPK models used to describe the pharmacokinetics of zearalenone after iv injection (model I) (A) and oral administration(model II) (B).

(A) (B)

VdC

dtQ

C

KQ

C

KQ

C

KQ

C

K

QC

K

VeVe

LiLi

LiKi

Ki

KiHe

He

HeTe

Te

Te

BrBr

B

= + + +

+rr

MuMu

MuAd

Ad

AdVe VeQ

C

KQ

C

KQ C+ + −

(1)

VdC

dtQ

C

KQ CAr

ArLu

Lu

LuVe Ve= − (2)

VdC

dtQ C Q

C

KT

TT Ar T

T

P = − (3)

VdC

dt(Q Q Q Q ) C Q

C

K

QC

KQ

C

LiLi

Li Sp St SI Ar SpSp

Sp

StSt

StSI

SI

= − − − +

+ +KK

QC

KCl

C

KSILi

Li

Liint

Li

Li − −

(4)

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1398 B. S. SHIN ET AL.

• For lung:

For oral administration, additional differential mass balanceequations were written for small intestine [Eq. (6)], conjugatedzearalenone compartment [Eq. (7)], fast absorption lumen[Eq. (8)], and slow absorption lumen [Eq. (9)]. The “fast”uptake from the lumen has no delay, whereas the five transitcompartments delay the “slow” uptake from lumen. Thisdelayed uptake produces the second peak.

• For small intestine:

• For the conjugated zearalenone compartment:

• For the fast absorption from lumen:

• For the slow absorption from lumen:

For these equations, VVe, VAr, VLu, VLi, VSp, VKi, VHe, VTe,VBr, VMu, VAd, VSt, VSI, VGlu, VLu,f, and VLu,s represent the vol-umes of vein, artery, lung, liver, spleen, kidneys, heart, testes,brain, muscle, adipose tissue, stomach, small intestine, conju-gated zearalenone compartment, fast absorption lumen com-partment, and slow absorption lumen compartment,respectively. CVe, CAr, CLu, CLi, CSp, CKi, CHe, CTe, CBr, CMu,CAd, CSt, CSI, CGlu, CLu,f, and CLu,s represent zearalenone

concentrations in vein, artery, lung, liver, spleen, kidneys,heart, testes, brain, muscle, adipose tissue, stomach, smallintestine, conjugated zearalenone compartment, fast absorptionlumen compartment, and slow absorption lumen compartment,respectively. QVe, QAr, QLu, QLi, QSp, QKi, QHe, QTe, QBr, QMu,QAd, QSt, and QSI represent the venous and arterial blood flowrates, and the blood flow rates of lung, liver, spleen, kidneys,heart, testes, brain, muscle, adipose tissue, stomach, and smallintestine, respectively. KLu, KLi, KSp, KKi, KHe, KTe, KBr, KMu,KAd, KSt, and KSI represent the tissue-to-blood partition coeffi-cients of lung, liver, spleen, kidneys, heart, testes, brain, mus-cle, adipose tissue, stomach, and small intestine, respectively.Clint represents the liver intrinsic clearance. Ka,f and Ka,s repre-sent the absorption rate constants of fast and slow absorptionfrom lumen compartments, respectively. Kre represents therecirculation rate constant. Ktr represents the transition rateconstant between slow absorption lumen compartments. Kel,f,Kel,s, and Kel,Glu represent the elimination rate constants of thefast absorption from lumen compartment, the slow absorptionfrom lumen compartment, and the conjugated zearalenonecompartment, respectively.

The tissue volumes and blood flow rates for 0.25-kg rat and70-kg man were obtained from the literature (Bernareggi &Rowland, 1991; Davies & Morris, 1993; Brown et al., 1997;Hosseini-Yeganeh & McLachlan, 2002). The liver intrinsicclearance (Clint) was estimated by computer fitting using invivo concentration–time data obtained from the iv bolus injec-tion study. The rate constants of Ka,f, Ka,s, Ktr, Kre, Kel,f, Kel,s,and Kel,Glu were estimated in Adapt II (see later discussion)using in vivo concentration–time data from the po administra-tion study. The steady-state tissue-to-blood partition coeffi-cients (KP) were determined from the iv infusion study. TheKP values were calculated by converting serum concentrationsto blood concentrations using the blood-to-serum partitioncoefficients.

The partition coefficient KLi for an eliminating tissue (liver,for example) is:

The partition coefficient KP for non-eliminating tissues (allother tissues) is:

Parameters were estimated by nonlinear regression using theADAPT II program with maximum likelihood estimation withadditive and proportional error (University of SouthernCalifornia). Model simulations were performed in BerkeleyMadonna (University of California) using the final parameterestimates.

VdC

dtQ C Q

C

KLu

LuLu Ve Lu

Lu

Lu = − (5)

VdC

dtQ C Q

C

KK C V

K C

SISI

SI Ar SI SI

SIa, f Lu, f Lm, f

a, s Lu, s

= − +

+ VVLm, s

(6)

VdC

dtBile Cl C (K K ) C VGlu

Gluint Li el, Glu re Glu Glu= − + (7)

VdC

dtINPUT K C V (K K ) C VLu, f

Lu, fre Glu Glu el, f a, f Lu, f Lu, f= + − + (8)

VdC

dtK C V K C V

VdC

Lu, sLu, s1

el, f Lu, f Lu, s tr Lu, s1 Lu, s

Lu, sLu

= −

,, s2tr Lu, s1 Lu, s tr Lu, s2 Lu, s

Lu, sLu, s3

tr

dtK C V K C V

VdC

dtK

= −

= C V K C V

VdC

dtK C V

Lu, s2 Lu, s tr Lu, s3 Lu, s

Lu, sLu, s4

tr Lu, s3

−

= LLu, s tr Lu, s4 Lu, s

Lu, sLu, s5

tr Lu, s4 Lu, s el

K C V

VdC

dtK C V (K

−

= − ,, s a, s Lu, s5 Lu, s+ K ) C V

(9)K

C

C

Q Cl

QLi

Li, SS

B, SS

Li Hepatic

Li= .

+(10)

KC

CP

T, SS

B, SS= (11)

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PBPK MODELING OF ZEARALENONE 1399

Model ValidationFor model prospective validation, rats (n = 4) received an iv

injection of 8 mg/kg zearalenone at 0, 3, and 6 h. During thedosing period, blood samples (0.3 ml each) were collected at 5,10, 15, and 30 min and 1, 2, 3, 3.083, 3.167, 3.25, 3.5, 4, 6,6.083, 6.25, and 7 h. Blood samples were centrifuged at 3500 × gfor 10 min. The harvested serum samples were kept at −20ºCuntil analysis. The rats were sacrificed at 1, 3.5, and 7 h follow-ing initiation of the multiple iv injections, and lung, spleen,liver, kidneys, heart, testes, brain, muscle, adipose tissue,stomach, and small intestine were collected and tissue weightswere recorded. The tissue samples were homogenized in phos-phate buffer (Tissue Tearor, Biospec Co., Bartlesville, OK)and kept at −20ºC until analysis.

For prospective model validation, zearalenone concentrationsobserved in blood and various tissues after multiple iv injectionswere compared with the model predicted concentration–timeprofiles. These concentrations were not used during estimationof PBPK parameters.

Extrapolation to HumansInitially, blood and tissue concentrations were simulated

in rat after once daily po administration of zearalenone (dose0.1 mg/kg) for 20 d. The maximum (Cmax) and minimum(Cmin) concentrations, time to reach Cmax (Tmax), and averageconcentrations (Cav) in blood and various tissues were pre-dicted. The Cav was calculated as AUC0-τ at steady statedivided by the dosing interval (24 h). For human extrapolation,

a daily po dose of zearalenone that yields an average steady-state blood concentration identical to that found in rat wasdetermined by modeling. The steady-state concentration–time profiles of zearalenone in blood and various tissues aswell as values for Cmax, Cmin, Tmax, and Cav were predicted fora 70-kg human.

RESULTSPhysiological parameter values of the blood flow rates,

tissue volumes, and steady-state tissue-to-blood partition coef-ficients (Kp) used in PBPK modeling are reported in Table 1.The liver intrinsic clearance, tissue-to-serum partition coeffi-cient, and transfer rate constants are shown in Table 2. Experi-mentally observed serum zearalenone concentrations andmodel simulated concentration–time profiles in rats after ivbolus injection and po administration (dose 8 mg) are shown inFigures 2 and 3, respectively. The proposed models adequatelydescribed the observed concentration–time data. After ivinjection, serum zearalenone concentrations declined multi-exponentially. The fractions of dose excreted unchanged were0.91 ± 0.64% in bile and 0.5 ± 0.2% in urine. This suggests thatzearalenone is primarily metabolized or eliminated asunchanged drug by the liver.

After po administration, a distinct secondary peak wasobserved in the serum concentration–time profile, whichwas absent after iv injection. The absolute oral bioavailability was2.7%. The fraction of po dose excreted unchanged in bile washigher than after iv injection (1.89 ± 0.89 vs. 0.91 ± 0.64 %).

TABLE 1 Physiological Parameters Used in PBPK Modeling (Models I and II)

Rat Human

Tissue VTa (ml) QT

a (ml/min) Kpb VT

a(ml) QTa (ml/min)

Arterial blood 11.3 43 3600 5240Venous blood 5.6 43 1800 5240Lung 2.1 43 2.31 ± 0.76 1170 5240Liver 10.3 11.8 4.56 ± 1.20 1690 1650Spleen 0.6 0.63 0.81 ± 0.18 192 77Kidneys 3.7 9.23 5.55 ± 1.86 310 1100Heart 1.2 3.92 1.16 ± 0.31 267 150Testes 2.5 0.45 0.49 ± 0.12 36 2.4Brain 1.2 1.33 1.06 ± 0.23 1450 700Muscle 122 7.5 0.43 ± 0.21 30,000 750Adipose 10 0.4 3.31 ± 1.26 10,000 260Stomach 1.1 1.14 1.30 ± 0.76 155 39Small intestine 11.4 7.52 37.80 ± 17.07 1650 1100

aTissue volumes (VT) and blood flow rates (QT) in 0.25-kg rat and 70-kg human were obtained from the literature (Bernareggi & Rowland, 1991; Davies & Morris, 1993; Brown et al., 1997; Hosseini-Yeganeh & McLachlan., 2002).

bTissue-to-blood partition coefficients (Kp) (mean ± SD) were calculated in rats after iv infusion to steady state.

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1400 B. S. SHIN ET AL.

The terminal elimination half-life (t1/2) was significantly pro-longed after po administration compared to after iv injection(16.8 ± 8.4 vs. 2.8 ±1.1 h). Other detailed information on ivand po absorption pharmacokinetics is reported elsewhere(Shin et al., 2009b).

Figure 4 shows the prospective model validation for mul-tiple iv injections of 8 mg/kg doses every 3 h. Simulatedconcentrations agreed well with observed concentrations inblood and various tissues. Subsequently, steady-statezearalenone concentration–time profiles were simulated forrat blood and tissues after once daily po exposures (0.1 mg/kg/d) (Figure 5). The average steady-state Cmax, Cmin, Tmax,and Cav in rat blood and tissues are reported in Table 3. Thehighest steady-state AUCo-τ and Cav values were found insmall intestine, followed by liver, kidneys, adipose, lung,

stomach, heart, and brain, and these values were higher thanthose found in blood.

For extrapolation to humans, a daily po dose of zearale-none needed to achieve an average steady-state blood con-centration identical to that found in rat (0.014 ng/ml) waspredicted to be 0.0312 mg/kg/d. The simulated time coursesof zearalenone concentrations in human blood and varioustissues after once daily po administration (dose 0.0312 mg/kg/d) are shown in Figure 6. The average steady-state Cmax,Cmin, Tmax, and Cav in human blood and tissues are reportedin Table 4.

DISCUSSIONThis study reports the development of PBPK models for

zearalenone and its application to interspecies extrapolationof pharmacokinetics from rats to human. The PBPK modelfor iv injection adequately described the concentration–timeprofiles in serum and various tissues in rats after a singleand multiple iv doses (Figures 2 and 4). Unlike iv injection,po administration of zearalenone showed a second peak inthe serum concentration–time profiles in both intact andBDC rats. The second peak phenomenon for zearalenonewas reported previously in rats and pigs after po administra-tion (Biehl et al., 1993; Mallis et al., 2003). The biliaryexcretion of zearalenone and its glucuronide conjugates andsubsequent enterohepatic recirculation may produce the sec-ond peak in rats (Shin et al., 2009, this issue). After poadministration of zearalenone in BDC rats, minor secondpeaks were found and the terminal half-life was longer com-pared to iv injections (Shin et al., 2009, this issue). Thissuggests that in addition to enterohepatic recirculation, a

TABLE 2 Estimates for the Intrinsic Clearance, Rate Constants, and the Blood-to-Serum Partition Coefficient Used in PBPK

Modeling (Model II)

Parameter Value

CLint (ml/min/kg0.75) 126.5Ka,fast (kg0.25/min) 0.010564Ka,slow (kg0.25/min) 0.000046Kel,slow (kg0.25/min) 0.002034Ktr (kg0.25/min) 0.030851Kel,fast (kg0.25/min) 1.320875Kel,glu (kg0.25/min) 0.000168Kre (kg0.25/min) 0.002251Blood-to-serum partition coefficient 0.67 ± 0.08

FIG. 2. Simulated (dotted line) and observed (❍) serum concentrations ofzearalenone in rats after iv injection (8 mg/kg) (n = 4).

Time (hr)

0 2 4 6 8 10 12

Seru

m Z

eara

leno

ne C

once

ntra

tion

(ng/

ml)

0.01

0.1

1

10

100

1000

10000

100000

FIG. 3. Simulated (dotted and solid lines) and observed serumconcentrations of zearalenone in intact (•, n = 6) and bile duct-cannulated (❍,n = 3) rats after oral administration (8 mg/kg).

Time (hr)

0 4 8 12 16 20 24

Seru

m Z

eara

leno

ne C

once

ntra

tion

(ng

/ml)

0.01

0.1

1

10

100

Intact (prediction)Bile removal (prediction)Intact (observation)Bile removal (observation)

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PBPK MODELING OF ZEARALENONE 1401

secondary absorption site might also contribute to the sec-ond peak phenomenon after po administration, since entero-hepatic recirculation is absent in BDC rats.

The PBPK model for po absorption was constructed toreflect the glucuronidation, biliary excretion, enterohepaticrecirculation, and fast (immediate) and slow (delayed)

absorption processes from the lumen compartment. The poabsorption model adequately described the secondary peak phe-nomenon and the post-distribution characteristics (Figure 3).Using this model, steady-state zearalenone concentrations inblood and tissues were simulated for rats after once daily poexposures (0.1 mg/kg/d) (Figure 5). The average steady-state

FIG. 4. Simulated (solid lines) and observed (•) concentrations of zearalenone in blood, lung, liver, spleen, kidneys, heart, testes, brain, muscle, adipose tissue,stomach, and small intestine after multiple iv injections (8 mg/kg) every 3 h in rats (n = 4).

Time (hr)

Blood

Liver

Lung

Spleen

Kidney Heart

0 3 6 9 12 15 18 21 24

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none

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cent

ratio

n (n

g/g)

1e–1

1e+0

1e+1

1e+2

1e+3

1e+4

1e+5

1e+6

Time (hr)0 3 6 9 12 15 18 21 24

0 3 6 9 12 15 18 21 24

0 3 6 9 12 15 18 21 240 3 6 9 12 15 18 21 24

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none

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cent

ratio

n (n

g/g)

1e–1

1e+0

1e+1

1e+2

1e+3

1e+4

1e+5

1e+6

1e+7

Time (hr)0 3 6 9 12 15 18 21 24

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ratio

n (n

g/g)

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10

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10000000

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1402 B. S. SHIN ET AL.

blood zearalenone concentration predicted in rat was 0.014ng/ml (Table 3). The oral dose of zearalenone used in thissimulation (0.1 mg/kg/d) is within the range of doses associ-ated with the lowest-observed-adverse-effect level (NOAEL )of 0.06 mg/kg/d and no-observed-effect level (NOEL) of 0.2mg/kg/d reported for reproductive effects in pubertal pigs asthe most sensitive species (Kuiper-Goodman et al., 1987;

Zinedine et al., 2007). A daily human oral dose required toachieve the same steady-state blood concentration found inrats (0.014 ng/ml) was predicted to be 0.0312 mg/kg/d (or2.18 mg/70 kg/d). Figure 6 shows the steady-state zearalenoneconcentration–time profiles in blood and tissues simulated forhuman after multiple oral exposures (dose 0.0312 mg/kg/d).The daily human oral intake used in human extrapolation

FIG. 4. (Continued).

Time (hr)

Testis Brain

Muscle Adipose

Stomach Small intestine

0 3 6 9 12 15 18 21 24

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none

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cent

ratio

n (n

g/g)

0.1

1

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Time (hr)0 3 6 9 12 15 18 21 24

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cent

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Time (hr)0 3 6 9 12 15 18 21 24

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cent

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PBPK MODELING OF ZEARALENONE 1403

(0.0312 mg/kg/d) is higher than the provisional maximumtolerable daily intake of 0.5 μg/kg/d proposed by the JointFAO/WHO Expert Committee on Food Additives estab-lished based on the NOAEL and NOEL values described

earlier (JECFA, 2000). The developed and prospectivelyvalidated PBPK models may be useful to predict humanblood and tissue concentrations of zearalenone in humansafter oral intake.

FIG. 5. Simulated time courses of zearalenone concentrations in rat blood and tissues after once daily oral administration (dose 0.1 mg/kg/d).

Time (hr)

0 48 96 144 192 240 288 336 384 432 480

Zea

rale

none

Con

cent

rati

on (

ng/m

l or

ng/g

)

0.0001

0.001

0.01

0.1

1

10

100

Small Intestine

Liver Kidney

Adipose Lung

StomachHeartBrain

Muscle

TestisSpleen

ArteryVein

TABLE 3 Pharmacokinetic Parameters of Zearalenone Predicted in Rat After Once Daily Oral Administration

(0.1 mg/kg/d) to Steady State

Organ Tmax (hr) Cmax,ss (ng/ml) Cmin,ss (ng/ml)AUC0-τ,ss(ng.hr/ml) Cav (ng/ml)

Arterial blood 0.33 0.033 0.007 0.33 0.014Venous blood 0.33 0.033 0.007 0.323 0.014Lung 0.33 0.077 0.016 0.752 0.031Liver 0.20 1.379 0.253 12.11 0.505Spleen 0.33 0.027 0.006 0.26 0.011Kidneys 0.37 0.183 0.038 1.80 0.075Heart 0.33 0.038 0.008 0.38 0.016Testes 0.40 0.016 0.003 0.16 0.007Brain 0.37 0.035 0.007 0.35 0.014Muscle 0.50 0.014 0.003 0.14 0.006Adipose 4.60 0.065 0.024 1.08 0.045Stomach 0.37 0.043 0.009 0.42 0.018Small intestine 0.03 19.681 3.131 150.54 6.273

Note. Parameter values were calculated based on steady-state concentration–time profiles simulated by PBPK modeling.

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1404 B. S. SHIN ET AL.

REFERENCESBaldwin, R. S., Williams, R. D., and Terry, M. K. 1983. Zeranol: A review of

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Jodlbauer, J., Zöllner, P., and Lindner, W. 2000. Determination of zeranol,taleranol, zearalenone, a- and b-zearalenol in urine and tissue by high-performance liquid chromatography-tandem mass spectrometry. Chro-matographia 51:681–687.

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FIG. 6. Simulated time courses of zearalenone concentrations in human blood and tissues after once daily oral administration (dose 0.0312 mg/kg/d).

Time (hr)0 48 96 144 192 240 288 336 384 432 480

Zea

rale

none

Con

cent

rati

on (

ng/m

l or

ng/g

)

0.0001

0.001

0.01

0.1

1

10

100

Small Intestine

Liver Kidney Adipose

Lung

StomachHeartBrain

MuscleTestis

Spleen

ArteryVein

TABLE 4 Pharmacokinetic Parameters of Zearalenone Predicted

in Humans After Once Daily Oral Administration (0.0312 mg/kg/d) to Steady State

OrganCmax,ss (ng/ml)

Cmin,ss (ng/ml)

AUC0-τ,ss (ng.hr/ml)

Cav (ng/ml)

Arterial blood 0.039 0.010 0.33 0.014Venous blood 0.039 0.010 0.33 0.014Lung 0.091 0.023 0.76 0.032Liver 0.873 0.175 5.87 0.245Spleen 0.032 0.008 0.27 0.011Kidneys 0.217 0.055 1.81 0.076Heart 0.045 0.011 0.38 0.016Testes 0.019 0.005 0.16 0.007Brain 0.042 0.010 0.35 0.014Muscle 0.015 0.010 0.14 0.006Adipose 0.072 0.034 1.08 0.045Stomach 0.050 0.013 0.43 0.018Small intestine 11.210 1.984 66.88 2.787

Note. Parameter values were calculated based on steady-state con-centration–time profiles simulated by PBPK modeling.

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fluorescence detection for the determination of zearalenone in rat serum.Chromatographia 69:295–259.

Shin, B. S., Hong, S. H., Hwang, S. W., Kim, H. J., Lee, J. B., Yoon, H. S.,Kim, D. J., and Yoo, S. D.2009b. Determination of zearalenone by liquidchromatography/tandem mass spectrometry and application to a pharmaco-kinetic study. Biomed. Chromatogr. 23: 1014–1021.

Shin, B. S., Hong, S. H., Bulitta, J. B., Hwang, S. W., Kim, H. J., Lee, J. B.,Yoon, H. S., Kim, D. J., and Yoo, S. D. 2009. Dose-linearity of pharmaco-kinetics, oral absorption and tissue distribution of zearalenone in rats. J.Toxicol. Environ. Health, this issue.

Songsermsakul, P., Sontag, G., Cichna-Markl, M., Zentek, J., and Razzazi-Fazeli, E. 2006. Determination of zearalenone and its metabolites in urine,plasma and faeces of horses by HPLC-APCI-MS. J. Chromatogr. B843:252–261.

Zinedine, A., Soriano, J. M., Moltó, J. C., and Ma1es, J. 2007. Review on thetoxicity, occurrence, metabolism, detoxification, regulation and intake ofzearalenone: an oestrogenic mycotoxin. Food Chem. Toxicol. 45:1–18.

Zöllinger, P., Jodlbauer, J., Kleinova, M., Kahlbacher, H., Kuhn, T.,Hochsteiner, W., and Linder, W. 2002. Concentration levels of zearale-none and its metabolites in urine, muscle tissue, and liver samples ofpigs fed with mycotoxin-contaminated oats. J. Agric. Food Chem.50:2494–2501.

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