Gm

JXMa

b

c

d

e

f

g

h

h

•••••

a

ARRAA

KAPPRPT

f

h0

Toxicology Letters 229 (2014) 41–51

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

estational and lactational exposure to the polychlorinated biphenylixture Aroclor 1254 modulates retinoid homeostasis in rat offspring

avier Estebana,b,1, Lubna E. Elabbasa,1, Daniel Borga, Maria Herlina, Agneta Åkessona,avier Barberc, Gerd Hamscherd, Heinz Naud, Wayne J. Bowerse,f, Jamie S. Nakaie,atti Vilukselag,h, Helen Håkanssona,∗

Institute of Environmental Medicine, Karolinska Institutet, Stockholm, SwedenInstituto de Bioingeniería, Universidad Miguel Hernández de Elche, Elche (Alicante), SpainCentro de Investigación Operativa, Universidad Miguel Hernández, Elche (Alicante), SpainInstitute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine, Hannover, GermanyNeurotoxicology Laboratory, Environmental Health Sciences and Research Bureau, Health Canada, Ottawa, CanadaNeuroscience Department, Carleton University, Ottawa, CanadaDepartment of Environmental Health, THL – National Institute for Health and Welfare, Kuopio, FinlandDepartment of Environmental Science, University of Eastern Finland, Kuopio, Finland

i g h l i g h t s

Gestational and lactational exposure to A1254 altered retinoid levels in liver, kidney and serum in rat dams and offspring.Hepatic retinoids were associated with hepatic EROD, PROD and BROD, serum T3 and T4 levels as well as body and liver weights.Both sexes showed similar retinoid alterations, which were the only observations still apparent at postnatal day 350.Dioxin-like (DL) congeners were considered responsible for most of the observed effects according to the A1254 DL activity.The results lend further support to the proposal by OECD to measure retinoids for identification of endocrine alterations.

r t i c l e i n f o

rticle history:eceived 19 December 2013eceived in revised form 24 April 2014ccepted 25 April 2014vailable online 2 June 2014

eywords:roclor 1254olychlorinated biphenyls (PCBs)erinatal exposureetinoidsartial least square regression

a b s t r a c t

Polychlorinated biphenyls (PCBs) induce a broad spectrum of biochemical and toxic effects in mammalsincluding alterations of the vital retinoid (vitamin A) system. The aim of this study was to characterizealterations of tissue retinoid levels in rat offspring and their dams following gestational and lactationalexposure to the PCB mixture Aroclor 1254 (A1254) and to assess the interrelationship of these changeswith other established sensitive biochemical and toxicological endpoints. Sprague-Dawley rat dams wereexposed orally to 0 or 15 mg/kg body weight/day of A1254 from gestational day 1 to postnatal day (PND)23. Livers, kidneys and serum were collected from the offspring on PNDs 35, 77 and 350. Tissue and serumretinoid levels, hepatic cytochrome P450 (CYP) enzymes and serum thyroid hormones were analyzed. Amultivariate regression between A1254 treatment, hepatic retinoid levels, hepatic CYP enzymes activities,thyroid hormone levels and body/liver weights was performed using an orthogonal partial least-squares(PLS) analysis. The contribution of dioxin-like (DL) components of A1254 to the observed effects was

oxic equivalency also estimated using the toxic equivalency (TEQ) concept. In both male and female offspring short-termalterations in tissue retinoid levels occurred at PND35, i.e. decreased levels of hepatic retinol and retinoicacid (RA) metabolite 9-cis-4-oxo-13,14-dihydro-RA with concurrent increases in hepatic and renal all-

trans-RA levels. Long-term changes consisted of decreased hepatic retinyl palmitate and increased renal retinol levels that were apparent until PND350. Retinoid system alterations were associated with alteredCYP enzyme activities and serum thyroid hormone levels as well as body and liver weights in both

∗ Corresponding author at: Institute of Environmental Medicine, Karolinska Institutet, P.O. Box 210, SE-171 77 Stockholm, Sweden. Tel.: +46 8 5248 7527;ax: +46 8 34 38 49.

E-mail address: Helen.Hakansson@ki.se (H. Håkansson).1 These authors contributed equally to this work.

ttp://dx.doi.org/10.1016/j.toxlet.2014.04.021378-4274/© 2014 Elsevier Ireland Ltd. All rights reserved.

42 J. Esteban et al. / Toxicology Letters 229 (2014) 41–51

offspring and dams. The estimated DL activity was within an order of magnitude of the theoretical TEQfor different endpoints, indicating significant involvement of DL congeners in the observed effects. Thisstudy shows that tissue retinoid levels are affected both short- and long-term by developmental A1254exposure and are associated with alterations of other established endpoints of toxicological concern.

1

tlrbctdstt

iscdnraSnt

cDiefetactlB

ocPht(atr(liApieal

g

. Introduction

Polychlorinated biphenyls (PCBs) are persistent organic con-aminants that have been widely used as e.g. heat transfer fluids,ubricants, dielectric fluids in transformers and capacitors, flameetardants and sealants (Giesy and Kannan, 1998). PCBs show slowiodegradation and bioaccumulate in biota due to their chemi-al stability and lipophilic properties (Safe, 1994) and althoughheir production and use have been banned in most countries forecades (Giesy and Kannan, 1998), PCBs are still present in closedources, such as electrical transformers, and hence they continueo be released into the environment (Schmidt, 2010) and continueo contribute to human and wildlife exposure.

PCBs induce a broad spectrum of biochemical and toxic effectsn humans and animals (ATSDR, 2000). Fetuses and infants are con-idered particularly sensitive to chemical exposure due to theirritical developmental periods (Makri et al., 2004). Studies in chil-ren have shown associations between PCB exposure and, e.g.eurotoxicity (Chen et al., 1992a; Grandjean and Landrigan, 2006),espiratory infections (Dallaire et al., 2006; Donaldson et al., 2010)nd impaired fetal growth and development (Dallaire et al., 2013;agiv et al., 2007). Animal studies have confirmed that PCBs causeeurotoxicity, immunotoxicity and reproductive/developmentaloxicity as well as a range of other effects (ATSDR, 2000).

Individual PCB congeners are from a toxicological perspectivelassified as either dioxin-like (DL) or non-dioxin-like (NDL). TheL PCBs consist of 12 planar congeners that bind with high affin-

ty to the aryl hydrocarbon receptor (AhR) and induce DL toxicffects. These congeners have been assigned toxic equivalencyactors (TEFs) (Van den Berg et al., 2006) which can be used tostimate toxic equivalencies (TEQ) of combined exposures usinghe prototype dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)s reference compound. The NDL PCBs consist of 197 non-planarongeners that bind with low affinity or not at all to the AhR andhus do not exhibit a typical DL toxicity pattern but may modu-ate other signaling pathways (Elabbas et al., 2013; Kretschmer andaldwin, 2005).

Several types of commercial PCB mixtures have been producedf which Aroclor 1254 (A1254) is one of the most common. A1254ontain about 54% chlorine by weight and consist of DL and NDLCB congeners (Kodavanti et al., 2001). In animal studies A1254ave produced short- and long-term toxic effects, including neuro-oxicity, immunotoxicity and developmental/reproductive toxicityBowers et al., 2004; Donaldson et al., 2010; Safe, 1990, 1994; Tilsonnd Kodavanti, 1998). However, few studies have investigated theoxicity of A1254 following perinatal exposure, in particular withegard to the retinoid (vitamin A) system. Morse and Brouwer1995) observed reduced growth and changes in hepatic retinoidevels in rat offspring until postnatal day (PND) 90 and changesn organ weights until PND21 following gestational exposure to1254. Chu et al. (2008) showed that A1254 induced growth sup-ression and changes in organ weights and biochemical parameters

n rat offspring until PND77 following gestational and lactationalxposure. One of the endpoints which showed a high effect sizend was still affected at PND350 was decreased hepatic retinoid

evels.

The retinoid system, which is vital for developmental pro-ramming and morphogenesis, embryonic and postnatal growth,

© 2014 Elsevier Ireland Ltd. All rights reserved.

immune function and reproduction (Blomhoff and Blomhoff, 2006)is under complex regulation by multiple enzymes, binding proteinsand hormonal receptors as briefly indicated in Fig. 1.

The diet-derived hormone precursors are stored in the liver andother tissues mainly as retinyl palmitate, and can, as needed, beconverted to its biologically and/or hormonally active forms, e.g.retinol, retinal, or retinoic acid (RA) (Balmer and Blomhoff, 2002).Transcriptional retinoid functions are mediated by all-trans-RAand its metabolite 9-cis-RA via the nuclear retinoic acid recep-tor (RAR) and retinoid X receptor (RXR) families (Balmer andBlomhoff, 2002). The all-trans-RA metabolite 9-cis-4-oxo-13,14-dihydro-retinoic acid (9c-4o-13,14-dh-RA), also an agonist of RAR(Schuchardt et al., 2009), is a sensitive marker to TCDD exposure(Fletcher et al., 2005; Schmidt et al., 2003a), environmental mix-tures (Elabbas et al., 2014) and a marker of dietary vitamin A intake(Schmidt et al., 2003b). Rodent studies have shown that alter-ation of tissue retinoid levels, i.e. reduction of hepatic retinoids andincrease of renal retinoids, are among the most sensitive endpointsfollowing exposure to dioxins and DL PCBs (Chen et al., 1992b; Chuet al., 2001; Fletcher et al., 2001, 2005; Håkansson et al., 1991a,b;Van Birgelen et al., 1994a,b). Functional consequences of a mod-ulated retinoid system during development are expected to beadverse and, hence, effects on the retinoid system has been con-sidered an important endpoint and proposed for inclusion intotesting guidelines for evaluating endocrine modulating propertiesof chemicals (OECD, 2012).

The aim of the present study was to further clarify alterationsof the retinoid system in rat offspring and their dams followinggestational and lactational exposure to A1254, with a focus on indi-vidual retinoid forms including the signaling forms all-trans-RA and9c-4o-13,14dh-RA, in liver, kidneys and serum. Also, differences inresponse between sex or between offspring and dams was evalu-ated and a partial least square (PLS) regression was performed toassess associations between alterations of tissue retinoid levels andalready established biochemical and toxicological endpoints fol-lowing A1254 exposure. It was also evaluated to which extent DLcomponents of A1254 were causing the observed effects by usingthe TEQ-concept for DL compounds.

2. Materials and methods

2.1. Chemicals

Technical grade A1254 (lot number 124–191, Accustandard Inc.-New Haven,CT) was dissolved in corn oil. All-trans-retinol, retinyl palmitate, retinyl acetate,all-trans-RA and acitetrin were purchased from Sigma. All solvents were at leastHPLC grade and obtained from Merck (Darmstadt, Germany) or Mallinckrodt Baker(Greisheim, Germany).

2.2. Animals, experimental design, and use of previously published data

All treatment procedures and housing conditions were conformed to the Cana-dian Council on Animal Care guidelines and approved by the Animal Care Committeeof Health Canada. Details of animal housing, experimental design, gross and clinicalobservations as well as pathology have been reported earlier in Chu et al. (2008).Briefly, pregnant female Sprague-Dawley rats received corn oil vehicle or 15 mgA1254/kg body weight (bw) from gestation day 1 until weaning on PND23. The dos-ing solution (1 �1 A1254/g bw) was administered onto Honey Graham crackers of

2 g of weight (Nabisco Ltd, Toronto, Canada) and provided as such to the dams. Bloodand tissues of dams were collected seven days after weaning, i.e. on PND30-32; whileblood and tissues from one male and one female rat offspring from each litter weresampled at PNDs 35, 77 and 350, respectively. Blood and tissue materials were kept

J. Esteban et al. / Toxicology Letters 229 (2014) 41–51 43

*

*

* UGTs

Retinoyl glucuronides

*

REOH Retinyl esters

RAL all-trans-RA

OH-/Oxo-RA REH

LRAT

ADHs RDHs RALDHs,

CYP 1A1, 1B1 CYP26,

CYP 2B, 3A

CRBP-REOH

CRABP-RA

REOH-RBP-TTR (in plasma)

9c-4o-13,14dh-RA 9-cis-RA

Development and growth

Metabolism and homeostasis Reproduction and vision

*

NRs RXR RAR RXR * *

Fig. 1. Retinoid metabolism, transport and gene regulation. Retinoic acid (RA) required for transcriptional activation of RAR-RXR heterodimers is usually produced fromretinol (REOH) in target cells. REOH is derived from dietary forms of vitamin A (e.g. retinyl esters or �-caroten). A tight control of RA levels in the cell is necessary to ensureproper gene regulatory function, e.g. during critical developmental stages. In the cell, retinol is bound to CRBP while RA is bound to CRABP. Catabolism of RA seems to involveseveral CYPs, many of which are induced by xenobiotics. Retinoids can also be glucuronidated by UGTs for excretion. REOH, retinol; RAL, retinal; RA, retinoic acid; REH,retinyl ester hydrolase; LRAT, lecithin:retinol acyltransferase; RBP-TTR, retinol binding protein-transthyretin-complex; CRBP, cellular retinol binding protein; ADH, cytosolicalcohol dehydrogenase; RDH, retinol dehydrogenase; RALDH, retinal dehydrogenase; CYP, cytochrome P450 superfamily; CRABP, cellular retinoic acid binding protein; RAR,r id hork . DashG 2), Ro

fhOsabs

2

arNerrwsl(w

baBpt(um2wL1

B

mdr

2

2

p

etinoic acid receptor; RXR, retinoid X receptor; NRs, nuclear receptors (e.g. thyronown to be affected by chemicals; →, biotransformation; −, binding equilibriumomaa et al. (2012), Nilsson and Håkansson (2002), Novák et al. (2008), OECD (201

rozen at −80 ◦C for subsequent analyses. Body and organ weights, serum thyroidormone levels and hepatic cytochrome P450 (CYP) activity data (ethoxyresorufin-deethylation (EROD), pentoxyresorufin O-dealkylation (PROD) and benzyloxyre-

orufin O-dealkylation (BROD)) are presented for all the time-points, i.e. PND35, 77nd 350 (see Section 2.4.1). PND35 data were previously presented in tabular formaty Chu et al. (2008) while PND77 and 350 data were mentioned in text only whenignificances were observed.

.3. Analyses of retinoids, CYP activities and thyroid hormones

Retinyl palmitate and retinol in livers and kidneys were analyzed for both sexest all time-points and dams by HPLC as described in Nilsson et al. (2000). Briefly,etinoids were extracted from tissues using diisopropyl ether and separated on aucleosil C18 5-�m HPLC column (Macherey-Nagel, GmbH, Germany) using anthanol:water (90:10, v/v) mobile phase and detected with a JASCO821-FP fluo-escence detector (�ex = 325 nm, �em = 475 nm). Samples for retinyl palmitate andetinol in serum, that were only available for female offspring at PND77 and dams,ere separated on a J’sphere ODS-H80 column (4.6 mm × 150 mm, 4-�m particle

ize) obtained from YMC (Schermbeck, Germany) and detected with an ultravio-et detector at 325 nm as described by Schmidt et al. (2003b). The limit of detectionLOD) for retinol was 60 ng/g tissue or 1.2 ng/ml serum. The LOD for retinyl palmitateas 70 ng/g tissue or 2.2 ng/ml serum.

All-trans-RA and 9c-4o-13,14dh-RA in livers and kidneys were analyzed foroth sexes at all time-points and for dams, and in serum for female offspringt PND77 and for dams by HPLC as previously described (Schmidt et al., 2003b).riefly, separation of all-trans-RA and 9c-4o-13,14dh-RA from retinol and retinylalmitate was achieved by solid-phase-extraction using an aminopropyl phase. All-rans-RA and 9c-4o-13,14dh-RA were then separated on a Spherisorb ODS2 column2.1 mm × 150 mm, 3 �m particle size) obtained from Waters (Eschborn, Germany)sing a binary gradient, and were detected with a UV/vis detector at 340 nm. Theetabolite, 9c-4o-13,14dh-RA, was extracted as described elsewhere (Schmidt et al.,

002). Since there is no available synthetic standard, levels of 9c-4o-13,14dh-RAere calculated using the calibration of 13-cis-4-OH-RA (Schmidt et al., 2002). The

OD for all-trans-RA was 0.2 ng/g tissue or 0.14 ng/ml serum. The LOD for 9c-4o-3,14dh-RA was 0.3 ng/g tissue or 0.21 ng/ml serum.

Hepatic CYP1A, CYP2B and CYP3A induction were assessed as EROD, PROD andROD activities as previously described (Chu et al., 2008).

Total serum thyroxine (T4) triiodothyronine (T3) and thyroid stimulating hor-one (TSH) levels were analyzed using commercial radioimmunoassay kits as

escribed in Chu et al. (2008) with LODs of 5.0 ng/ml, 0.2 ng/ml and 1.0 ng/ml,espectively.

.4. Data evaluation

.4.1. Statistical analysisVariables showed a normal distribution according to Kolmogorov–Smirnov non-

arametric tests and Q–Q plots. Thus, data were described by mean ± standard

mone receptor); UGT, UDP-glucuronosyltransferase; *Indicates enzymes/proteinsed lines indicate uncertainties in current knowledge. For overview references seeos et al. (2011), and Zile (2001).

deviation (SD). Values for any sample that was below LOD were set to LOD/2. Dif-ferences between exposure groups of female and male offspring at PNDs 35, 77and 350 as well as of dams at PND30 were tested against corresponding controlsusing two-tailed independent-samples T test. The level of significance was set at a p-value of <0.05. All statistical analyses were performed using IBM SPSS 18.0 StatisticalSoftware (IBM Corporation, New York).

2.4.2. PLS analysisA multivariate regression between A1254 exposure, body and liver weight,

serum T3 and T4, hepatic EROD, PROD and BROD activities (variables namedgenerally as endpoints from now on) and the hepatic levels of all-trans-RA, 9c-4o-13,14dh-RA, retinol and retinyl palmitate (retinoid variables from now on) wasperformed by PLS analysis (Bastien et al., 2005; Eriksson et al., 2006; Höskuldsson,1988; Wold et al., 2001) using R software version 2.9.2 (R Development Core Team,R Foundation for Statistical Computing, Vienna, Austria). Females and males wereconsidered as two different experiments to assess sex-dependent effects exerted bytreatment. Thus, one PLS method was built for female rats at PNDs 35, 77, and 350,and for dams, which provided four different effects estimates, respectively. AnotherPLS method was applied for male rats at PNDs 35, 77 and 350. Variables within com-ponents were entered in a PLS model using a backward method. The final model hadthe smallest number of components with similar explained variance of significantvariables among models by monitoring the (equivalent) root-mean-square error(RMS).

RMS =

√√√√n∑i

(yi − yi)2

n

where (yi − yi) was the difference between the predicted and the true value, andwhere n is the number of predictions (Mevik and Cederkvist, 2004). The PLS-2 mul-tivariate response model allowed high explained variances of both endpoints andretinoid variables as determined by R2X and R2Y fractions, respectively. A modelwith both R2X and R2Y values >0.6 was considered to be acceptable in order to studyassociations between variables. A variable importance in the projection (VIP) value≥0.8 in, at least, one component and time point was considered a cutoff for enter-ing significant variables in the PLS model (Chong and Jun, 2005). VIP values werecomputed as follows:

VIPj =

√√√√p

h∑(SS(bktk)(wjk/ � wk �)

2/

h∑SS(bktk)

k=1 k=1

SS(bktk) = b2kttktk

Endpoints (X) and retinoid variables (Y) were visualized on correlation circlesusing their correlations with PLS components.

4 ogy Le

wte7oa(maAtmwP

a

2

wtdd22sppc

TCSa

DL

4 J. Esteban et al. / Toxicol

The PLS method was applied for offspring at PNDs 35, 77 and 350 as well as dams,hich described the time-course effects of A1254 on hepatic retinoid levels by using

wo components. The applied PLS models explained 91% and 88% of the variance ofndpoints (X) in female and male offspring at PND35, 87% of variance for dams,3% and 62% of variance for female and male offspring at PND77, and 67% and 57%f variance for female and male offspring at PND350 (Supplementary Table 1). Thepplied PLS models explained 83% and 73% of the variance in retinoid concentrationsY) of female and male offspring at PND35, 81% in dams, 60% and 51% in female and

ale offspring at PND77, and 59% and 58% of variance for female and male offspringt PND350 (Supplementary Table 1). The models with two components described1254-related hepatic retinoid alterations in offspring at PND35 and dams better

han they did in offspring at PNDs 77 and 350 (Supplementary Table 1). In fact, theodels were optimized to fit A1254-related effects (Supplementary Table 1), whichere apparent in offspring at PND35 and dams, while A1254 effects were subtle at

NDs 77 and 350.Supplementary material related to this article can be found, in the online version,

t http://dx.doi.org/10.1016/j.toxlet.2014.04.021.

.4.3. Calculation of TCDD equivalents of A1254To estimate the DL activity of A1254 changes in retinoid levels in this study as

ell as changes in body and organ weights and thyroid hormone levels were quan-itatively compared to the effect of TCDD on these measures from a separate TCDDose–response study using the same rat strain (Sprague Dawley) with a similar studyesign as the present study (Håkansson, H., personal communication; Finnilä et al.,010; Giese et al., 2007) using the procedure previously described (Elabbas et al.,

011). Briefly, the TCDD equivalents were calculated as the dose which caused theame effect size (%) in the dose–response relationships of the TCDD study as in theresent study at 15 mg A1254/kg bw. The TCDD equivalents of A1254 were com-ared to the theoretical daily TEQ of A1254, which was derived from the chemicalomposition of A1254 described in Elabbas et al. (2011). The TEQ was calculated to

able 1oncentrations of retinol, retinyl palmitate, all-trans-retinoic acid, and 9-cis-4-oxo-13prague Dawley offspring at postnatal days (PNDs) 35, 77 and 350 and dams at PND30 fot PND23 at daily doses of 0 and 15 mg/kg bw/day.

Endpoints Females

N/group 0 mg/kg

Hepatic retinyl palmitate (�mol/g)Offspring at PND35 9–13 0.36 ± 0.04Offspring at PND77 9–13 1.4 ± 0.2Offspring at PND350 9–13 5.4 ± 0.9Dams at PND30 9–13 2.9 ± 0.3

Hepatic retinol (nmol/g)Offspring at PND35 9–13 13 ± 3Offspring at PND77 9–13 18 ± 4Offspring at PND350 9–13 96 ± 66Dams at PND30 9–13 30 ± 4

Hepatic all-trans-retinoic acid (pmol/g)Offspring at PND35 5 15 ± 1Offspring at PND77 5 23 ± 11Offspring at PND350 5 26 ± 8Dams at PND30 5 41 ± 8

Hepatic 9c-4o-13,14dh-retinoic acid (pmol/g)Offspring at PND35 5 15 ± 5Offspring at PND77 5 30 ± 7Offspring at PND350 5 46 ± 12Dams at PND30 5 35 ± 5

Renal retinol (nmol/g)Offspring at PND35 9–13 3.9 ± 0.9Offspring at PND77 9–13 2.8 ± 0.3Offspring at PND350 9–13 2.2 ± 0.4Dams at PND30 9–13 3.8 ± 0.6

Renal all-trans-retinoic acid (pmol/g)Offspring at PND35 5 11 ± 1Offspring at PND77 5 8.9 ± 1.5Offspring at PND350 5 8.5 ± 0.6Dams at PND30 5 9.1 ± 0.7

ata are expressed as mean ± SD. Blank = not applicable. LOD = Limit of detection. RetinylOD.

* p < 0.05 significantly different from control according to two-tailed independent-sam** p ≤ 0.01 significantly different from control according to two-tailed independent-sam

*** p ≤ 0.001 significantly different from control according to two-tailed independent-sa

tters 229 (2014) 41–51

be 0.12 �g/kg bw/day by using the TEQ concept for DL compounds (Van den Berget al., 2006).

3. Results

3.1. Hepatic retinoids

Hepatic retinyl palmitate levels were decreased in female off-spring at PNDs 35, 77 and 350 by 67%, 20% and 30%, respectively(Table 1). Similarly, hepatic retinyl palmitate levels were reducedin male offspring by 53%, 21% and 31% at PNDs 35, 77 and 350,respectively (Table 1). Hepatic retinol levels were reduced in femaleand male offspring at PND35, by 54% and 50%, respectively, but nosignificant changes were observed in offspring at PNDs 77 or 350(Table 1). Hepatic all-trans-RA levels were increased at PND 35 infemale and male offspring by 60% and 43%, respectively, but werenot altered at PNDs 77 and 350 (Table 1). Hepatic 9c-4o-13,14dh-RAlevels in female and male offspring at PND35 were reduced by93% and 70%, respectively, though the reductions did not remainat PNDs 77 or 350 (Table 1).

In dams, both hepatic retinyl palmitate level and hepatic retinol

levels were decreased by 32% and 33%, respectively, at PND30(Table 1). The hepatic levels of all-trans-RA in the dams wasdecreased by 59% and its metabolite 9c-4o-13,14-dh-RA was belowthe LOD, thus reduced by >97% (Table 1).

,14-dihydro-retinoic acid (9c-4o-13,14-dh-retinoic acid) in livers and kidneys ofllowing maternal oral Aroclor 1254 from gestation day 1 until the end of lactation

Males

15 mg/kg 0 mg/kg 15 mg/kg

0.12 ± 0.02*** 0.32 ± 0.04 0.15 ± 0.03***

1.1 ± 0.1** 1.0 ± 0.1 0.81 ± 0.08***

3.8 ± 0.6*** 4.2 ± 0.6 2.9 ± 0.5***

2.0 ± 0.3***

6.2 ± 1.1 *** 12 ± 2 6.2 ± 1.1***

15 ± 4 13 ± 3 12 ± 460 ± 19 59 ± 17 43 ± 1520 ± 5***

24 ± 6* 14 ± 1 20 ± 5*

15 ± 4 16 ± 4 14 ± 622 ± 14 35 ± 12 51 ± 1517 ± 2**

1.0 ± 0.8*** 12 ± 5 3.6 ± 1.5*

22 ± 7 16 ± 7 10 ± 447 ± 10 33 ± 4 27 ± 9<LOD***

12 ± 5*** 4.4 ± 1.1 10 ± 5.5**

3.6 ± 0.8* 4.9 ± 1.1 7.2 ± 2.0**

3.4 ± 1.5* 3.3 ± 0.9 4.9 ± 1.1**

5.8 ± 2.6

14 ± 1** 8.9 ± 2.7 22 ± 148.1 ± 0.5 6.0 ± 1.5 10 ± 2*

7.2 ± 0.7 4.8 ± 1.3 5.4 ± 2.08.6 ± 0.7

palmitate and 9c-4o-13,14-dh-RA in kidneys of offspring and dams were below the

ples T test.ples T test.

mples T test.

ogy Letters 229 (2014) 41–51 45

3

o3w7inrwl(w

ops

3

eert

Acpfli(

3

aPasittgcm

fj

wdfaaaP

svwnam

0

0.8

LPR

OD

DO

SE

LB

RO

D

T4

LE

RO

D

T3

LIW

E

BW

0

0.8

DO

SE

T4

LE

RO

D

T3

LIW

E

LB

RO

D

LPR

OD

BW

0.0

0.8

DO

SE

LE

RO

D

LPR

OD

LB

RO

D

BW

T4

T3

LIW

E

A

B

C

VIP

V

IP

VIP

Fig. 2. Variable importance in the projection (VIP) by component of endpointsentered in the partial least square (PLS) regression analysis for hepatic retinoidconcentrations in (A) female and (B) male offspring at PNDs 35 and (C) damsat termination. Dose, Aroclor 1254; BW, body weight; Liwe, liver weight; EROD,Ethoxyresorufin-O-deethylase enzyme activity; PROD, hepatic 7-pentoxyresorufinO-dealkylase enzyme activity; BROD, hepatic benzyloxyresorufin O-dealkylase; T3,concentrations of circulating triidothyronine; T4, concentrations of circulating thy-roxine. Solid bar, component t1 and open bar component t2. Variables with variableimportance in the projection (VIP) values higher than 0.8 in, at least, one compo-

J. Esteban et al. / Toxicol

.2. Renal retinoids

Renal retinol levels were increased in female offspring through-ut the time-course of the study by 208%, 29% and 54% at PNDs5, 77 and 350, respectively (Table 1). Similarly, renal retinol levelsere increased in male offspring by 136%, 47% and 48% at PNDs 35,

7 and 350, respectively (Table 1). Renal all-trans-RA levels werencreased at PND35 by 27% in females and by 146% in males, thoughot statistically significant in the males (p = 0.062). At PND77, theenal all-trans-RA level was increased by 73% in male offspring,hile it was not affected in females at PND77. Renal all-trans-RA

evels were not affected in male or female offspring at PND 350Table 1). Renal retinyl palmitate and 9c-4o-13,14-dh-RA levelsere below the LODs in the offspring (data not shown).

No treatment-related changes were observed for renal retinolr all-trans-RA levels in dams (Table 1) and the levels of retinylalmitate and 9c-4o-13,14-dh-RA were below the LODs (data nothown).

.3. Serum retinoids

In offspring, serum retinoid levels of controls and A1254-xposed rats did not differ significantly at PND77, i.e. retinol lev-ls were 541 ± 127 and 576 ± 178 pmol/mL, respectively, those ofetinyl palmitate 78 ± 75 and 64 ± 37 pmol/mL, respectively, andhose of all-trans-RA 1.4 ± 0.4 and 1.5 ± 0.7 pmol/mL, respectively.

In dams, the serum retinol level was decreased in the1254-exposed group (338 ± 68 pmol/mL) by 29% (p < 0.01) whenompared to the control group (476 ± 43 pmol/mL). Serum retinylalmitate levels of control and A1254-exposed groups did not dif-er, i.e.78 ± 75 and 64 ± 37 pmol/mL, respectively. The all-trans-RAevel in serum of A1254-exposed dams (8.0 ± 1.6 pmol/mL) wasncreased by 196% (p < 0.001) when compared to control dams2.7 ± 1.5 pmol/mL).

.4. Associations between retinoid levels and other endpoints

In addition to changes in retinoid levels, both sexes of offspringnd dams also showed A1254-induced alterations in hepatic EROD,ROD and BROD activities, serum thyroid hormone levels as wells body and organ weights (Supplementary Figs. 1–6, partly pre-ented in Chu et al., 2008) and thus these endpoints were includedn the PLS-analysis. As previously reported by Chu et al. (2008)here was no impact of A1254 on litter size or sex ratios. However,here was a quantitative increase in mortality in the A1254-treatedroup. Nevertheless, nonparametric analysis indicated no signifi-ant differences between groups for either litter mortality or puportality.Supplementary material related to this article can be

ound, in the online version, at http://dx.doi.org/10.1016/.toxlet.2014.04.021.

For the applied PLS model, R2x and R2y values correspondedith the selection criteria for male and female offspring and theams (Supplementary Table 1). In both multivariate models foremale and male offspring, A1254 treatment, hepatic EROD, PRODnd BROD activities, body and liver weights, as well as serum T3nd T4 levels were considered to be significant endpoints (Fig. 2 And B), with the exceptions of body weight in female offspring atND35 (Fig. 2A) and serum T3 levels in dams (Fig. 2C).

The circle of correlations obtained by PLS shows the relation-hip between endpoints (see Section 2.4.2 PLS analysis) and retinoidariables (Fig. 3). The axes of the circle of correlations correspond

ith components t1 and t2. Both components are linear combi-ations of the endpoints (X) entered in the model, for which thessociation between endpoints (X) and retinoid variables (Y) is opti-ized. The importance of the endpoints (X) in each component is

nent, sex and time point were considered significant variables and entered in thePLS model. Females and males were considered different experiments to evaluatesex differences.

described with VIPs values (Fig. 2). Both endpoints (X) and retinoidvariables (Y) are visualized on the correlation circles using theircorrelations with the PLS components t1 and t2, i.e., the cosinesof the angles formed by the vector-variables in the plane give anindication of the correlations between the original variables.

The circle of correlation has a similar meaning as in the linearcorrelations of Pearson, but includes information about the inter-relationship of the variables as they are organized in the quadrants.Opposed quadrants represent opposed responses of the studied

variables. By convention, the retinoid reductions were plotted inthe upper right hand quadrant, for which all variables located inthe abscissa to the right of the main component t1 were reduced byA1254, whereas those in the abscissa to the left were increased by

4 ogy Le

Aitfb

ARawPpt

Fa4ec

6 J. Esteban et al. / Toxicol

1254. Similarly, in the secondary component t2, variables locatedn upper side of the ordinate are reduced whereas those located inhe lower side are increased. The variables which are located farrom the origin are those which were altered with a higher efficacyy A1254.

Induced hepatic EROD, PROD and BROD activities following1254 treatment were associated with increased hepatic all-trans-A concentrations (cluster of variables with projections in thebscissa to the left of component t1 of Fig. 3A and B). There

as an inverse relationship between the cluster of induced EROD,

ROD and BROD activities and reduced hepatic retinol, retinylalmitate, and 9c-4o-13,14-dh-RA concentrations, for which thosewo clusters of variables, i.e. induced activities (left) and reduced

−1.0 −0.5 0.0

−1.

0−

0.5

0.0

0.5

1.0

Circle of Correlat

X−compon

X−

com

pone

nt t

2

DOSE

LBROD

LPROD

LEROD

T3

C Dams

−1.0 −0.5 0.0 0.5 1.0

−1.

0−

0.5

0.0

0.5

1.0

Circle of Correlations on t1, t2

X−component t1

X−

com

pone

nt t

2

DOSE

LBROD

LPROD

LEROD

LIWE

BW

T3

T4

LCREP

LCATR

LCREO

LC9C4

A Female offspirng

ig. 3. Partial least square regression circle of correlations on X-components t1 and t2 for rt termination by hepatic EROD, PROD and BROD inductions, thyroid hormones, body and l-oxo-13,14-dihydro-retinoic acid; LCREO, retinol; LCREP, retinyl palmitate. Dose, Aroclonzyme activity; PROD, 7-pentoxyresorufin O-dealkylase enzyme activity; BROD, benzyoncentrations of circulating thyroxine. Females and males were considered different exp

tters 229 (2014) 41–51

retinoids (right), showed symmetric projections in component t1of Fig. 3A and B. Furthermore, hepatic retinoid concentration alter-ations were associated with circulating T3 and T4 levels (clusterof variables with similar projections in component t1 of Fig. 3Aand B). A1254-related reductions of body weights at PND35 wereassociated with 9c-4o-13,14-dh-RA reductions observed in males(similar projections in both components t1 and t2 of Fig. 3B) andwith reductions of both retinyl palmitate and the 9c-4o-13,14-dh-RA in females (Fig. 3A), though low VIP values of body weight were

found in female offspring at PND35. Both retinyl palmitate and9c-4o-13,14-dh-RA of female offspring at PND35 responded in asimilar manner (similar projections in both components t1 and t2of Fig. 3A).

0.5 1.0

ions on t1, t2

ent t1

LIWE

BWT4

LCREP

LCATR

LCREO

LC9C4

−1.0 −0.5 0.0 0.5 1.0

−1.

0−

0.5

0.0

0.5

1.0

Circle of Correlations on t1, t2

X−component t1

X−

com

pone

nt t

2

DOSE

LBROD

LPROD

LEROD

LIWE

BW

T4

T3

LCREO

LCREP

LC9C4

LCATR

B Male offspring

etinoid concentrations of (A) female and (B) male offspring at PNDs 35 and (C) damsiver weight and Aroclor 1254 treatment. LCATR, all-trans-retinoic acid; LC9C4, 9-cis-r 1254; BW, body weight; Liwe, liver weight; EROD, Ethoxyresorufin-O-deethylaseloxyresorufin O-dealkylase; T3, concentrations of circulating triidothyronine; T4,eriments to evaluate sex differences.

J. Esteban et al. / Toxicology Le

Fig. 4. Calculation of TCDD equivalents on the basis of hepatic 9-cis-4-oxo-13,14-dihydro-retinoic acid (pmol/g) reduction in male offspring at PND35following perinatal exposure to Aroclor1254. The solid curve represents the fitteddose–response model and the dashed line indicates the benchmark dose of 0.1 TCDDet

ijrt(Iwpo

3

setrrat1(2mic

4

Aaoh

quivalents (�g/kg bw), which was calculated at a reduction of 70% as observed inhe Aroclor1254-treated male offspring at PND35.

In dams, there was an inverse relationship between the cluster ofnduced EROD, PROD and BROD activities by A1254 treatment (pro-ections in the abscissa to the left of component t1 of Fig. 3C) and theeduced hepatic retinoid concentrations, for which those two clus-ers of variables, i.e. induced activities (left) and reduced retinoidsright), showed symmetric projections in component t1 of Fig. 3C.mportantly, both body weight and T4 circulating level reductions

ere associated with all-trans-RA, 9c-4o-13,14-dh-RA and retinylalmitate concentration reductions (projections in component t1f Fig. 3C).

.5. TCDD equivalents of A1254

The theoretical calculated total DL activity of A1254 corre-ponded to a TEQ of 5.3 �g/kg bw (Table 2). The estimated TCDDquivalents (See Section 2.4.3) for hepatic retinyl palmitate reduc-ions were 3.3 and 3.1 �g/kg bw for PND35 females and males,espectively, which was 62% and 58%, respectively, of the theo-etical A1254 TEQ (Table 2). The estimated TCDD equivalent forll-trans-RA increases was 1.4 �g/kg bw for males at PND35, 26% ofhe theoretical A1254 TEQ. Estimated TCDD equivalents for 9c-4o-3,14dh-RA reductions were 0.35 and 0.10 �g/kg bw for femalesFig. 4) and males at PND35, respectively, corresponding to 7% and% of the theoretical A1254 TEQ, respectively (Table 2). The esti-ated TCDD equivalents for changes in serum T4 levels as well as

n body and thymus weights were between 1.3 and 2.0 �g/kg bw,orresponding to 25–38% of the theoretical A1254 TEQ (Table 2).

. Discussion

This study showed that gestational and lactational exposure to

1254 altered retinoid levels in liver, kidney and serum in rat damsnd their offspring. These changes were associated with alterationsf established endpoints, which responded to A1254 exposure, i.e.epatic EROD, PROD and BROD activities, serum T3 and T4 levels

tters 229 (2014) 41–51 47

as well as body and liver weights. Although the magnitude of theretinoid system alterations decreased with time, effects on retinoidlevels were, in contrast to the other endpoints, still apparent atPND350. The estimated DL activity was in the same order of mag-nitude as the theoretical TEQ for different endpoints indicating thatDL congeners were significantly involved in the observed effects.

4.1. Effects of A1254-exposure on retinoid levels

The mammalian embryo and neonate acquire its vitamin A fromthe maternal circulation and breast milk (Båvik et al., 1996; Debierand Larondelle, 2005; Håkansson et al., 1987; Morse and Brouwer,1995), however, the complete retinoid system machinery requiredto synthesize the functional and signaling retinoid forms as neededis in place already at the earliest stages of development (Ulven et al.,2000). Fetal and early postnatal vitamin A levels are very low, and itis not until after weaning that the retinoid storage forms increaserapidly, i.e. when the offspring start eating vitamin A-containingdiet (Håkansson et al., 1987). It is well known that functionallyactive retinoid forms such as retinol, retinal and all-trans-RA areunder strict control by multiple retinoid system specific enzymesand binding proteins (Fig. 1) in terms of tissue levels as well as lev-els in circulation (Novák et al., 2008). It is only under circumstancessuch as clear dietary vitamin A-excess and deficiency situations,and possibly also under chemical stress situations, that these lev-els of functional retinoid forms are violated (Novák et al., 2008).Retinyl ester levels which contribute to a high extent to the totalhepatic vitamin A levels, in contrast, can vary substantially depend-ing on dietary conditions (Green and Green, 1994; Tan et al., 2014)and without obvious functional consequences.

We observed both short- and long-term effects on tissueretinoid levels in offspring following A1254 exposure and with sim-ilar responses in both sexes. The short-term effects, apparent atPND35, were manifested as decreases in hepatic retinol and 9c-4o-13,14dh-RA levels and increased hepatic and renal all-trans-RAlevels. Furthermore, hepatic levels of 9c-4o-13,14dh-RA, which hassimilar transcriptional activity as all-trans-RA both in vitro andin vivo (Schuchardt et al., 2009), was reduced in the A1254-exposedoffspring on PND35. Because all-trans-RA is believed to mediatemost of the transcriptional retinoid functions, it can be assumed,that the increased hepatic levels of all-trans-RA found at PND 35in the offspring after A1254 exposure of the dams, could indicate amodulation of the retinoid system, which is compatible with thereduced growth (Chu et al., 2008) and altered bone parameters(Elabbas et al., 2011) that have previously been reported from thesame animal experiment. The PLS analyses of the present study lendfurther support to this hypothesis.

The long-term effects of gestational and lactational exposure toA1254 were evident as decreased hepatic retinyl palmitate levelsand increased renal retinol levels throughout the study and werestill evident at PND350. These long-term changes may be related tothe relatively long half-life of the PCB congeners in A1254 (Elabbaset al., 2011), ranging from 54 to 124 days (Öberg et al., 2002). Thefact that these effects remained throughout the study indicate apermanent change in retinoid storage and/or mobilization (Greenand Green, 1994; Kelley et al., 2000), which might reflect that theorganism responds to the chemical insult by a dynamic attempttoward rescue and stabilization of the all-trans-RA homeostasis inorder to maintain vital cell and body functions.

The transient nature of other investigated endpoints in thepresent animal experiment, i.e. CYP enzyme inductions, changesin thyroid hormone levels along with other biochemical endpoints

and histopathological findings (Chu et al., 2008), as well as changesin bone geometry, density and biomechanical properties (Elabbaset al., 2011) support such an hypothesis. However, additionaldynamic tissue changes, such as bone remodeling, which were not

48 J. Esteban et al. / Toxicology Letters 229 (2014) 41–51

Table 2Estimation of TCDD equivalents of Aroclor 1254 to modulate hepatic retinyl palmitate, all-trans-retinoic acid and 9-cis-4-oxo-13,14-dihydro-retinoic acid concentrations,serum thyroxine, and body and thymus weights of Sprague-Dawley rat offspring on postnatal day 35 as compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Endpoint Females Males

Aroclor 1254effect size (%)

Estimated TCDDequivalenta

(�g/kg bw)

Comparison tocalculated TEQb (%)

Aroclor 1254effect size (%)

Estimated TCDDequivalenta

(�g/kg bw)

Comparison tocalculated TEQb (%)

Hepatic retinyl palmitate −67 3.3 62 −53 3.1 58Hepatic all-trans-retinoic

acidN.A. N.A. N.A. 45 1.4 26

Hepatic9c-4o-13,14-dihydroretinoic acid

−93 0.35 7 −70 0.10 2

Serum thyroxine N.A. N.A. N.A. −83 1.3 25Body weight −22 1.8 34 −18 2.0 38Thymus weight −40 1.6 30 −33 1.5 28

Hepatic retinoic acid did not show any dose–response relationship in females in the TCDD study at PND35 and hepatic retinol did not show any dose–response relationshipin the TCDD study in females nor in males, thus these endpoints are not included in the analysis. N.A. = not applicable.

a TCDD equivalents for different endpoints were calculated using dose response curves on TCDD from a study using a similar study design (Finnilä et al., 2010; Giese et al.,2007; Håkansson H, personal communication).

ty of t

apctu

aiBplewrslwwwtatg

As9crocilianc

4A

at

b TEQ, the toxic equivalent was considered to be the total 2,3,7,8-TCDD-like activi

ddressed in this study, might contribute to an alternative inter-retation of the observed retinoid system changes. Furthermore,ontinuous rather than intermittent exposure situations, such ashose humans experience, might contribute to yet an alternativenderstanding of these data.

Retinoid system results presented in this study are in goodgreement with other developmental/reproductive toxicity stud-es in rats (Elabbas et al., 2014; Kransler et al., 2007; Morse androuwer, 1995; Van der Ven et al., 2009). Decreased hepatic retinylalmitate and retinol levels have been reported in offspring fol-

owing gestational and lactational exposure to TCDD (Håkanssont al., 1987; Kransler et al., 2007, Morse and Brouwer, 1995), asell as hexacyclododecane (Van der Ven et al., 2009), or a human-

elevant mixture of organochlorines (Elabbas et al., 2014). In thetudy by Elabbas et al. (2014) an increase in hepatic all-trans-RAevels was reported in male offspring, but not in female offspring

hereas in the present study, where a 5-fold higher TEQ-dose levelas applied, hepatic all-trans-RA and 9c-4o-13,14-dh-RA levelsere affected in both male and female offspring suggesting that

here is no real difference in the type of response between malesnd females following the exposure to the organochlorine mix-ure or A1254. However, a sensitivity issue may remain betweenenders.

There were similarities and differences in the responses to1254 between offspring and dams. Both the offspring and damshowed decreased levels of hepatic retinyl palmitate, retinol, andc-4o-13,14dh-RA and increased levels of renal retinol, which isonsistent with results presented by Elabbas et al. (2014) fromats that were exposed during early development to a mixturef organochlorines, which mimic the Arctic exposure situation. Inontrast, the hepatic and renal levels of all-trans-RA were decreasedn the dams but increased in the offspring. Also, the all-trans-RAevel was elevated in serum of the dams whereas it was unaffectedn the offspring. These data need to be confirmed in future studiess there are no data to compare with at present. A possible expla-ation to the difference in response between dams and offspringould be due to differences in CYP induction patterns.

.2. Association between retinoids and other endpoints following1254 exposure

Results of the multivariate PLS regression showed a strongssociation between levels of hepatic retinyl palmitate, retinol, all-rans-RA, and 9c-4o-13,14dh-RA with serum levels of T3 and T4 in

he Aroclor 1254 dose of 15 mg/kg bw/day, which was estimated to be 5.3 �g/kg bw.

female and male offspring at PND35. These associated alterationsare in good agreement with observations in other rodent stud-ies analysing the retinoid and thyroid hormone systems followingA1254 exposure (Hallgren et al., 2001; Morse et al., 1996; Murket al., 1991). Both the retinoid and thyroid hormone results follow-ing the A1254 exposure should be understood in the light of manyreports on interactions between the RXR, RAR, and TR on the onehand, and AhR, CAR and PXR signaling pathways on the other hand(Andreola et al., 1997; Brtko and Dvorak, 2011; Nishimura et al.,2005) and on the level of thyroid hormone and retinol transportby the transthyretin-retinol-binding protein complex in the blood(Novák et al., 2008).

EROD activity was strongly associated with the increase of all-trans-RA levels in offspring. The increased hepatic all-trans-RA inthe offspring was strongly associated with increased hepatic EROD-activity, which is compatible with CYP1A1 (Tomita et al., 1996) andCYP1B1 (Chambers et al., 2007; Chen et al., 2000; Choudhary et al.,2004) induced all-trans-RA synthesis. In the dams, on the otherhand, the decreased hepatic all-trans-RA levels, were strongly asso-ciated with increased hepatic PROD and BROD-activities, which iscompatible with increased degradation of all-trans-RA via CYP2Band CYP3A (Chen et al., 2009). The inductions of EROD, PROD andBROD activities, liver enlargement and body weight reductions inthis study, which were associated with reduced hepatic retinol andretinyl palmitate levels are in line with findings from other stud-ies in rodent offspring following A1254 exposure (Chu et al., 2005,2008; Morse and Brouwer, 1995).

Under the situation described in the present work, alteredgrowth might be related with the retinoid modulation in targettissues, as PLS showed associations between body weight andretinoid concentrations. In the liver, the increased production ofretinoic acid in offspring might be related with the observed hepa-tomegaly (Perea et al., 2000), though liver enlargement is oftenobserved after induction of metabolizing enzymes and presenceof hypertrophy as observed after exposure to TCDD (Hakanssonet al., 1987) and A1254 (Chu et al., 2005, 2008; Morse and Brouwer,1995).

To our knowledge, this is the first study to report an associa-tion between hepatic 9c-4o-13,14dh-RA levels and inductions ofhepatic EROD, PROD and BROD activities, liver enlargement and

body weight reductions in offspring and their dams. Offspring anddams were similar with regard to the association of retinoids withother endpoints, however, with the exception that serum T3 levelswere not important in the PLS model of the dams.

ogy Le

4

tt12ewpAosecitipAeopPm

tamT(tircth

4

teariTwaotpip

C

T

f

J. Esteban et al. / Toxicol

.3. Dioxin-like activity of A1254

TEQ calculations have previously been performed to estimatehe TCDD-like activity of A1254 using TEFs and have shown thathe DL PCB congeners 126, 118 and 105 contribute 46%, 29%, and2%, respectively, to the total theoretical A1254 TEQ (Elabbas et al.,011). The TCDD equivalents calculated in the present study forffects on hepatic retinoids, serum T4 levels, body and thymuseights ranged between 0.35 and 3.3 �g/kg bw, and were com-ared to the theoretical TEQ of 5.3 �g/kg bw for A1254 (Table 2).lthough somewhat less than expected, this range is still within anrder of magnitude of the theoretical TEQ value and hence con-idered to be acceptable estimations for most of the endpointsvaluated in this study. This finding suggests that it is mainly the DLongeners in A1254 that are responsible for the observed effects,ndicating that the A1254-induced alterations of the retinoid sys-em are mediated via activation of the AhR. In fact, the estimatednvolvement of DL components in alterations of hepatic retinylalmitate levels were similar to a corresponding estimate for1254-induced bone alterations (Elabbas et al., 2011). One possiblexplanation to the somewhat lower calculated TCDD equivalents aspposed to the theoretical TEQ in the present study could be theossibility of counteracting effects by one or several of the NDLCB components of the mixture, since the main constituents of theixture are NDL PCBs (ATSDR, 2000).From animal studies it is well established that the retinoid sys-

em is affected by dioxins and DL compounds (Reviewed in Nilssonnd Håkansson, 2002) and that AhR can play an important role inediating these effects (Nishimura et al., 2005; Okey et al., 1994).

here are differences in sensitivity to AhR ligands between speciesOkey et al., 2005) and the human AhR has less affinity for TCDDhan the rodent AhR (Van den Berg et al., 2006). However, theres considerable variability in sensitivity among humans in theiresponse to TCDD (U.S. EPA, 2004; Van den Berg et al., 2006) and,onsequently, AhR-mediated effects in rodents, such as effects onhe retinoid system, should therefore be considered relevant foruman health.

.4. Summary and conclusions

This single dose, time-course, study provides detailed informa-ion on alterations of the retinoid system in rat offspring followingxposure to A1254 during early development. The observed alter-tions were similar in both sexes and both short- and long-termetinoid system changes were observed. The estimated DL activ-ty of A1254 was within an order of magnitude of the theoreticalEQ for the different endpoints indicating that DL componentsere significantly involved in the observed retinoid system alter-

tions, which in turn were associated with alterations in endpointsf concern for the assessment of developmental toxicology. Takenogether the results presented herein lend further support to theroposal by OECD (2012) to include retinoid system analyses as an

mportant endpoint for identification and testing of chemicals withotential endocrine system modulating properties.

onflicts of interest statement

The authors declare that there are no conflicts of interest.

ransparency document

The Transparency document associated with this article can beound in the online version.

tters 229 (2014) 41–51 49

Acknowledgements

We thank Dr. Ih Chu, Environmental Health Sciences andResearch Bureau, Health Canada, Ottawa, Canada, for generouslyproviding the tissue material; Norbert Giese, Filip Rendel andChristina Trossvik for their technical assistance. This study wassupported by funds from the Commission of the European Com-munities projects BoneTox (EU-QLK-CT-2002-02528) CASCADE(FOOD-CT-2004-506319), and ATHON (FOOD-CT-2005-022923),Northern Contaminants Program, DIAND, Health Canada, Gen-eralitat Valenciana (BEST/2007/082 and BEST/2008/137, Spain),Programa José Castillejo (JC2007-00107, Ministerio de Educacióny Ciencia, Spain) as well as grants from Karolinska Institutet andthe Swedish Research Council/Medicine.

References

Andreola, F., FernandezSalguero, P.M., Chiantore, M.V., Petkovich, M.P., Gonzalez, F.J.,De Luca, L.M., 1997. Aryl hydrocarbon receptor knockout mice (AHR−/−) exhibitliver retinoid accumulation and reduced retinoic acid metabolism. Cancer Res.57, 2835–2838.

Agency for Toxic Substances and Disease Registry (ATSDR), 2000. Toxicological pro-file for polychlorinated biphenyls (PCBs). U.S. Department of Health and HumanServices, Available at: http://www.atsdr.cdc.gov/toxprofiles/tp17.pdf

Balmer, J., Blomhoff, R., 2002. Gene expression regulation by retinoic acid. J. LipidRes. 43, 1773–1808.

Bastien, P., Vinzi, V.E., Tenenhaus, M., 2005. PLS generalised linear regression. Com-put. Stat. Data Anal. 48, 17–46.

Blomhoff, R., Blomhoff, H.K., 2006. Overview of retinoid metabolism and function. J.Neurobiol. 66, 606–630.

Bowers, W.J., Nakai, J.S., Chu, I., Wade, M.G., Moir, D., Yagminas, A., Gill, S., Pulido, O.,Meuller, R., 2004. Early developmental neurotoxicity of a PCB/organochlorinemixture in rodents after gestational and lactational exposure. Toxicol. Sci. 77,51–62.

Brtko, J., Dvorak, Z., 2011. Role of retinoids, rexinoids and thyroid hormone in theexpression of cytochrome P450 enzymes. Curr. Drug Metab. 12, 71–88.

Båvik, C., Ward, S.J., Chambon, P., 1996. Developmental abnormalities in culturedmouse embryos deprived of retinoic by inhibition of yolk-sac retinol bindingprotein synthesis. Proc. Natl. Acad. Sci. U.S.A. 93, 3110–3114.

Chambers, D., Wilson, L., Maden, M., Lumsden, A., 2007. RALDH-independent gener-ation of retinoic acid during vertebrate embryogenesis by CYP1B1. Development134, 1369–1383.

Chen, Y.C., Guo, Y.L., Hsu, C.C., 1992a. Cognitive development of children prenatallyexposed to polychlorinated biphenyls (Yu-Cheng children) and their siblings. J.Formos. Med. Assoc. 91, 704–707.

Chen, L.C., Berberian, I., Koch, B., Mercier, M., Azais-Braesco, V., Glauert, H.P., Chow,C.K., Robertson, L.W., 1992b. Polychlorinated and polybrominated biphenyl con-geners and retinoid levels in rat tissues: structure–activity relationships. Toxicol.Appl. Pharmacol. 114, 47–55.

Chen, H., Howald, W.N., Juchau, M.R., 2000. Biosynthesis of all-trans-retinoic acidfrom all-trans-retinol: catalysis of all-trans-retinol oxidation by human P-450cytochromes. Drug Metab. Dispos. 28, 315–322.

Chen, P.J., Padgett, W.T., Moore, T., Winnik, W., Lambert, G.R., Thai, S.F., Hester, S.D.,Nesnow, S., 2009. Three conazoles increase hepatic microsomal retinoic acidmetabolism and decrease mouse hepatic retinoic acid levels in vivo. Toxicol.Appl. Pharmacol. 234, 143–155.

Chong, I., Jun, C., 2005. Performance of some variable selection methods when mul-ticollinearity is present. Chemom. Intell. Lab. Syst. 78, 103–112.

Choudhary, D., Jansson, I., Stoilov, I., Sarfarazi, M., Schenkman, J.B., 2004. Metabolismof retinoids and arachidonic acid by human and mouse cytochrome P450 1b1.Drug Metab. Dispos. 32, 840–847.

Chu, I., Lecavalier, P., Håkansson, H., Yagminas, A., Valli, V.E., Poon, P., Feeley, M.,2001. Mixture effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and polychlori-nated biphenyl congeners in rats. Chemosphere 43, 807–814.

Chu, I., Bowers, W., Caldwell, D., Nakai, J., Pulido, O., Yagminas, A., Wade, M., Moir,D., Gill, S., Mueller, R., 2005. Toxicological effects of gestational and lactationalexposure to a mixture of persistent organochlorines in rats: systemic effects.Toxicol. Sci. 88, 645–655.

Chu, I., Bowers, W.J., Caldwell, D., Nakai, J., Wade, M.G., Yagminas, A., Li, N., Moir,D., Elabbas, L., Håkansson, H., Gill, S., Mueller, R., Pulido, O., 2008. Toxicologicaleffects of in utero and lactational exposure of rats to a mixture of environmen-tal contaminants detected in Canadian Arctic human populations. J. Toxicol.Environ. Health A 71, 93–108.

Dallaire, F., Dewailly, E., Vézina, C., Muckle, G., Weber, J.P., Bruneau, S., Ayotte, P.,2006. Effect of prenatal exposure to polychlorinated biphenyls on incidence ofacute respiratory infections in preschool Inuit children. Environ. Health Per-

spect. 114, 1301–1305.

Dallaire, R., Dewailly, E., Ayotte, P., Forget-Dubois, N., Jacobson, S.W., Jacobson, J.L.,Muckle, G., 2013. Exposure to organochlorines and mercury through fish andmarine mammal consumption: Associations with growth and duration of ges-tation among Inuit newborns. Environ. Int. 54, 85–91.

5 ogy Le

D

D

E

E

E

E

F

F

F

G

G

G

G

G

H

H

H

H

HK

K

K

K

M

M

0 J. Esteban et al. / Toxicol

ebier, C., Larondelle, Y., 2005. Vitamins A and E: metabolism, roles and transfer tooffspring. Br. J. Nutr. 93, 153–174.

onaldson, S.G., Van Oostdam, J., Tikhonov, C., Feeley, M., Armstrong, B., Ayotte, P.,Boucher, O., Bowers, W., Chan, L., Dallaire, F., Dallaire, R., Dewailly, E., Edwards,J., Egeland, G.M., Fontaine, J., Furgal, C., Leech, T., Loring, E., Muckle, G., Nancar-row, T., Pereg, D., Plusquellec, P., Potyrala, M., Receveur, O., Shearer, R.G., 2010.Environmental contaminants and human health in the Canadian Arctic. Sci. TotalEnviron. 408, 5165–5234.

labbas, L.E., Herlin, M., Finnilä, M.A., Rendel, F., Stern, N., Trossvik, C., Bowers, W.J.,Nakai, J., Tuukkanen, J., Viluksela, M., Heimeier, R.A., Åkesson, A., Håkansson, H.,2011. In utero and lactational exposure to Aroclor 1254 affects bone geometry,mineral density and biomechanical properties of rat offspring. Toxicol. Lett. 207,82–88.

labbas, L.E., Westerholm, E., Roos, R., Halldin, K., Korkalainen, M., Viluksela, M.,Håkansson, H., 2013. Non dioxin-like polychlorinated biphenyl: exposure andhealth hazards. In: Rose, M., Fernandes, A. (Eds.), Persistent Organic Pollutantsand Toxic Metals in Foods. Woodhead Publishing Ltd., Cambridge (UK), pp.215–260.

labbas, L.E., Esteban, J., Barber, X., Bowers, W.J., Nakai, J., Hamscher, G., Nau, H.,Herlin, M., Åkesson, A., Borg, D., Håkansson, H., 2014. In utero and lactationalexposure to a mixture of environmental contaminants detected in CanadianArctic human populations alters retinoid levels in rat offspring with low marginsof exposure. J. Toxicol. Environ. Health A 77, 223–245.

riksson, L., Andersson, P.L., Johansson, E., Tysklind, M., 2006. Megavariate analysisof environmental QSAR data. Part I – A basic framework founded on principalcomponent analysis (PCA), partial least squares (PLS), and statistical moleculardesign (SMD). Mol. Divers. 10, 169–186.

innilä, M.A., Zioupos, P., Herlin, M., Miettinen, H.M., Simanainen, U., Håkans-son, H., Tuukkanen, J., Viluksela, M., Jamsa, T., 2010. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on bone material properties. J. Biomech.43, 1097–1103.

letcher, N., Hanberg, A., Håkansson, H., 2001. Hepatic vitamin a depletion is a sen-sitive marker of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in fourrodent species. Toxicol. Sci. 1, 166–175.

letcher, N., Giese, N., Schmidt, C., Stern, N., Lind, P.M., Viluksela, M., Tuomisto,J.T., Tuomisto, J., Nau, H., Håkansson, H., 2005. Altered retinoid metabolismin female long-evans and Han/Wistar rats following long-term 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treatment. Toxicol. Sci. 86, 264–272.

iese, N., Viluksela, M., Håkansson, H., Nau, H., 2007. 9-cis-4-oxo-13,14-dihydro-retinoic acid (9c-4o-dh-RA) – a new sensitive biomarker for TCDD toxicity.Toxicologist 96 (1), 834.

iesy, J., Kannan, K., 1998. Dioxin-like and non-dioxin-like toxic effects of polychlo-rinated biphenyls (PCBs): implications for risk assessment. Crit. Rev. Toxicol. 28,511–569.

omaa, M.S., Bridgens, C.E., Illingworth, N.A., Veal, G.J., Redfern, C.P., Brancale, A.,Armstrong, J.L., Simons, C., 2012. Novel retinoic acid 4-hydroxylase (CYP26)inhibitors based on a 3-(1H-imidazol- and triazol-1-yl)-2,2-dimethyl-3-(4-(phenylamino)phenyl)propyl scaffold. Bioorg. Med. Chem. 20, 4201–4207.

randjean, P., Landrigan, P.J., 2006. Developmental neurotoxicity of industrial chem-icals. Lancet 368, 2167–2178.

reen, M.H., Green, J.B., 1994. Vitamin A intake and status influence retinol balance,utilization and dynamics in rats. J. Nutr. 124, 2477–2485.

åkansson, H., Waern, F., Ahlborg, U., 1987. Effects of 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) in the lactating rat on maternal and neonatal vitamin astatus. J. Nutr. 117, 580–586.

åkansson, H., Johansson, L., Manzoor, E., Ahlborg, U.G., 1991a. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the vitamin A status of Hartley guineapigs, Sprague-Dawley rats, C57Bl/6 mice, DBA/2 mice, and Golden Syrian ham-sters. J. Nutr. Sci. Vitaminol. (Tokyo) 37, 117–138.

åkansson, H., Manzoor, E., Ahlborg, U.G., 1991b. Interaction between dietary vita-min A and single oral doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) onthe TCDD-induced toxicity and on the vitamin A status in the rat. J. Nutr. Sci.Vitaminol. (Tokyo) 37, 239–255.

allgren, S., Sinjari, T., Håkansson, H., Darnerud, P.O., 2001. Effects of polybromi-nateddiphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) on thyroidhormone and vitamin A levels in rats and mice. Arch. Toxicol. 75, 200–208.

öskuldsson, A., 1988. PLS regression methods. J. Chemom. 2, 211–228.elley, S.K., Nilsson, C.B., Green, M.H., Green, J.B., Håkansson, H., 2000. Mobilization

of vitamin A stores in rats after administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin: a kinetic analysis. Toxicol. Sci. 55, 478–484.

odavanti, P.R., Kannan, N., Yamashita, N., Derr-Yellin, E.C., Ward, T.R., Burgin, D.E.,Tilson, H.A., Birnbaum, L.S., 2001. Differential effects of two lots of aroclor 1254:congener-specific analysis and neurochemical end points. Environ. Health Per-spect. 109, 1153–1161.

ransler, K.M., Tonucci, D.A., McGarrigle, B.P., Napoli, J.L., Olson, J.R., 2007.Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters retinoidhomeostasis in maternal and perinatal tissues of the Holtzman rat. Toxicol. Appl.Pharmacol. 224, 29–38.

retschmer, X.C., Baldwin, W.S., 2005. CAR and PXR: xenosensors of endocrine dis-rupters? Chem. Biol. Interact. 155, 111–128.

akri, A., Goveia, M., Balbus, J., Parkin, R., 2004. Children’s susceptibility to chemi-

cals: a review by developmental stage. J. Toxicol. Environ. Health B. Crit. Rev. 7,417–435.

evik, B., Cederkvist, H., 2004. Mean squared error of prediction (MSEP) estimatesfor principal component regression (PCR) and partial least squares regression(PLSR). J. Chemom. 18, 422–429.

tters 229 (2014) 41–51

Morse, D.C., Brouwer, A., 1995. Fetal, neonatal, and long-term alterations in hepaticretinoid levels following maternal polychlorinated biphenyl exposure in rats.Toxicol. Appl. Pharmacol. 131, 175–182.

Morse, D.C., Wehler, E.K., Wesseling, W., Koeman, J.H., Brouwer, A., 1996. Alter-ations in rat brain thyroid hormone status following pre- and postnatal exposureto polychlorinated biphenyls (Aroclor 1254). Toxicol. Appl. Pharmacol. 136,269–279.

Murk, A.J., Vandenberg, J.H.J., Koeman, J.H., Brouwer, A., 1991. The toxicity of tetra-chlorobenzyltoluenes (Ugilec 141) and polychlorobiphenyls (Aroclor 1254 andPcb-77) compared in Ah-responsive and Ah-nonresponsive mice. Environ. Pol-lut. 72, 57–67.

Nilsson, C., Hoegberg, P., Trossvik, C., Azais-Braesco, V., Blaner, W., Fex, G., Harrison,E., Nau, H., Schmidt, C., van Bennekum, A., Håkansson, H., 2000. 2,3,7,8-Tetrachlorodibenzo-P-dioxin increases serum and kidney retinoic acid levelsand kidney retinol esterification in the rat. Toxicol. Appl. Pharmacol. 169,121–131.

Nilsson, C., Håkansson, H., 2002. The retinoid signaling system – a target in dioxintoxicity. Crit. Rev. Toxicol. 32, 211–232.

Nishimura, N., Yonemoto, J., Miyabara, Y., Fujii-Kuriyama, Y., Tohyama, C., 2005.Altered thyroxin and retinoid metabolic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin in aryl hydrocarbon receptor-null mice. Arch. Toxicol. 79, 260–267.

Novák, J., Benísek, M., Hilscherová, K., 2008. Disruption of retinoid transport,metabolism and signaling by environmental pollutants. Environ. Int. 34,898–913.

Öberg, M., Sjödin, A., Casabona, H., Nordgren, I., Klasson-Wehler, E., Håkansson, H.,2002. Tissue distribution and half-lives of individual polychlorinated biphenylsand serum levels of 4-hydroxy-2,3,3,4,5-pentachlorobiphenyl in the rat. Toxicol.Sci. 70, 171–182.

OECD, 2012. Detailed review paper on the state of the science on novel in vitro andin vivo screening and testing methods and endpoints for evaluating endocrinedisruptors. ENV/JM/MONO, pp. 23, Available at: http://search.oecd.org/officialdocuments/displaydocumentpdf/?cote=env/jm/mono%282012%2923&doclanguage=en

Okey, A.B., Riddick, D.S., Harper, P.A., 1994. The Ah receptor: mediator of the toxicityof 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Toxicol.Lett. 70, 1–22.

Okey, A.B., Franc, M.A., Moffat, I.D., Tijet, N., Boutros, P.C., Korkalainen, M., Tuomisto,J., Pohjanvirta, R., 2005. Toxicological implications of polymorphisms in recep-tors for xenobiotic chemicals: the case of the aryl hydrocarbon receptor. Toxicol.Appl. Pharmacol. 207, 43–51.

Perea, G., Salar, A., Altés, A., Brunet, S., Sierra, J., 2000. Acute hepatomegalywith severe liver toxicity due to all-trans-retinoic acid. Haematologica 85,551–552.

Roos, R., Andersson, P.L., Halldin, K., Hakansson, H., Westerholm, E., Hamers, T., Ham-scher, G., Heikkinen, P., Korkalainen, M., Leslie, H.A., Niittynen, M., Sankari, S.,Schmitz, H.J., van der Ven, L.T., Viluksela, M., Schrenk, D., 2011. Hepatic effectsof a highly purified 2,2′ ,3,4,4′ ,5,5′-heptachlorbiphenyl (PCB 180) in male andfemale rats. Toxicology 284, 42–53.

Safe, S., 1990. Polychlorinated-biphenyls (PCBs), dibenzo-para-dioxins (PCDDs),dibenzofurans (PCDFs), and related-compounds – environmental and mechanis-tic considerations which support the development of toxic equivalency factors(TEFs). Crit. Rev. Toxicol. 21, 51–88.

Safe, S., 1994. Polychlorinated-biphenyls (PCBs) – environmental-impact, biochem-ical and toxic responses, and implications for risk assessment. Crit. Rev. Toxicol.24, 87–149.

Sagiv, S.K., Tolbert, P.E., Altshul, L.M., Korrick, S.A., 2007. Organochlorine exposuresduring pregnancy and infant size at birth. Epidemiology 18, 120–129.

Schmidt, C.K., Volland, J., Hamscher, G., Nau, H., 2002. Characterization of a newendogenous vitamin A metabolite. Biochim. Biophys. Acta 1583, 237–251.

Schmidt, C.K., Hoegberg, P., Fletcher, N., Nilsson, C.B., Trossvik, C., Håkansson,H., Nau, H., 2003a. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters theendogenous metabolism of all-trans-retinoic acid in the rat. Arch. Toxicol 77,371–383.

Schmidt, C.K., Brouwer, A., Nau, H., 2003b. Chromatographic analysis of endogenousretinoids in tissues and serum. Anal. Biochem. 315, 36–48.

Schmidt, C., 2010. How PCBs are like grasshoppers. Environ. Sci. Technol. 44, 2752.Schuchardt, J.P., Wahlström, D., Rüegg, J., Giese, N., Stefan, M., Hopf, H., Pongratz,

I., Håkansson, H., Eichele, G., Pettersson, K., Nau, H., 2009. The endogenousretinoid metabolite S-4-oxo-9-cis-13,14-dihydro-retinoic acid activates retinoicacid receptor signalling both in vitro and in vivo. FEBS J. 276, 3043–3059.

Tan, L., Wray, A.E., Green, M.H., Ross, A.C., 2014. Retinol kinetics in unsupplementedand vitamin A-retinoic acid supplemented neonatal rats: a preliminary model.J. Lipid Res. 55 (6), 1077–1086, http://dx.doi.org/10.1194/jlr.M045229.

Tilson, H., Kodavanti, P., 1998. The neurotoxicity of polychlorinated biphenyls. Neu-rotoxicology 19, 517–525.

Tomita, S., Okuyama, E., Ohnishi, T., Ichikawa, Y., 1996. Characteristic properties ofa retinoic acid synthetic cytochrome P-450 purified from liver microsomes of3-methylcholanthrene-induced rat. Biochim. Biophys. Acta 1290, 273–281.

Ulven, S.M., Gundersen, T.E., Weedon, M.S., Landaas, V.O., Sakhi, A.K., Fromm, S.H.,Geronimo, B.A., Moskaug, J.O., Blomhoff, R., 2000. Identification of endogenousretinoids, enzymes, binding proteins, and receptors during early postimplanta-

tion development in mouse: important role of retinal dehydrogenase type 2 insynthesis of all-trans-retinoic acid. Dev. Biol. 220, 379–391.

United States Environmental Protection Agency (U.S. EPA), 2004. Exposureand Human Health Reassessment of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin(TCDD) and Related Compounds National Academy Sciences (NAS). Part II,

ogy Le

V

Vstudy in the rat. Toxicol. Appl. Pharmacol. 127, 209–221.

J. Esteban et al. / Toxicol

Chapter 2. Review Draft, Available at: http://www.epa.gov/ncea/pdfs/dioxin/nas-review/pdfs/part2/dioxin pt2 ch02 dec2003.pdf

an den Berg, M., Birnbaum, L.S., Denison, M., De Vito, M., Farland, W., Feeley, M.,Fiedler, H., Håkansson, H., Hanberg, A., Haws, L., Rose, M., Safe, S., Schrenk, D.,Tohyama, C., Tritscher, A., Tuomisto, J., Tysklind, M., Walker, N., Peterson, R.E.,2006. The 2005 World Health Organization reevaluation of human and mam-malian toxic equivalency factors for dioxins and dioxin-like compounds. Toxicol.Sci. 93, 223–241.

an der Ven, L.T., van de Kuil, T., Leonards, P.E., Slob, W., Lilienthal, H., Litens, S.,Herlin, M., Håkansson, H., Cantón, R.F., van den Berg, M., Visser, T.J., van Loveren,H., Vos, J.G., Piersma, A.H., 2009. Endocrine effects of hexabromocyclododecane(HBCD) in a one-generation reproduction study in Wistar rats. Toxicol. Lett. 185,51–62.

tters 229 (2014) 41–51 51

Van Birgelen, A., Vanderkolk, J., Fase, K., Bol, I., Poiger, H., Vanderberg, M., Brouwer,A., 1994a. Toxic potency of 2,3,3′ ,4,4′ ,5-hexachlorobiphenyl relative to and incombination with 2,3,7,8-tetrachlorodibenzo-P-dioxin in a subchronic feedingstudy in the rat. Toxicol. Appl. Pharmacol. 126, 202–213.

Van Birgelen, A., Vanderkolk, J., Fase, K., Bol, I., Poiger, H., Brouwer, A., Vandenberg,M., 1994b. Toxic potency of 3,3′ ,4,4′ ,5-pentachlorobiphenyl relative to and incombination with 2,3,7,8-tetrachlorodibenzo-P-dioxin in a subchronic feeding

Wold, S., Trygg, J., Berglund, A., Antti, H., 2001. Some recent developments in PLSmodeling. ChemometricsIntellig. Lab. Syst. 58, 131–150.

Zile, M.H., 2001. Function of vitamin A in vertebrate embryonic development. J. Nutr.131, 705–708.