Toxicology Letters 229 (2014) 41–51

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Gestational and lactational exposure to the polychlorinated biphenyl mixture Aroclor 1254 modulates retinoid homeostasis in rat offspring Javier Esteban a,b,1 , Lubna E. Elabbas a,1 , Daniel Borg a , Maria Herlin a , Agneta Åkesson a , Xavier Barber c , Gerd Hamscher d , Heinz Nau d , Wayne J. Bowers e,f , Jamie S. Nakai e , Matti Viluksela g,h , Helen Håkansson a,∗ a

Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Instituto de Bioingeniería, Universidad Miguel Hernández de Elche, Elche (Alicante), Spain c Centro de Investigación Operativa, Universidad Miguel Hernández, Elche (Alicante), Spain d Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine, Hannover, Germany e Neurotoxicology Laboratory, Environmental Health Sciences and Research Bureau, Health Canada, Ottawa, Canada f Neuroscience Department, Carleton University, Ottawa, Canada g Department of Environmental Health, THL – National Institute for Health and Welfare, Kuopio, Finland h Department of Environmental Science, University of Eastern Finland, Kuopio, Finland b

h 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.

a r t i c l e

i n f o

Article history: Received 19 December 2013 Received in revised form 24 April 2014 Accepted 25 April 2014 Available online 2 June 2014 Keywords: Aroclor 1254 Polychlorinated biphenyls (PCBs) Perinatal exposure Retinoids Partial least square regression Toxic equivalency

a b s t r a c t Polychlorinated biphenyls (PCBs) induce a broad spectrum of biochemical and toxic effects in mammals including alterations of the vital retinoid (vitamin A) system. The aim of this study was to characterize alterations of tissue retinoid levels in rat offspring and their dams following gestational and lactational exposure to the PCB mixture Aroclor 1254 (A1254) and to assess the interrelationship of these changes with other established sensitive biochemical and toxicological endpoints. Sprague-Dawley rat dams were exposed 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 serum retinoid levels, hepatic cytochrome P450 (CYP) enzymes and serum thyroid hormones were analyzed. A multivariate 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 also estimated using the toxic equivalency (TEQ) concept. In both male and female offspring short-term alterations in tissue retinoid levels occurred at PND35, i.e. decreased levels of hepatic retinol and retinoic acid (RA) metabolite 9-cis-4-oxo-13,14-dihydro-RA with concurrent increases in hepatic and renal alltrans-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 altered CYP 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; fax: +46 8 34 38 49. E-mail address: [email protected] (H. Håkansson). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.toxlet.2014.04.021 0378-4274/© 2014 Elsevier Ireland Ltd. All rights reserved.

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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 TEQ for different endpoints, indicating significant involvement of DL congeners in the observed effects. This study shows that tissue retinoid levels are affected both short- and long-term by developmental A1254 exposure and are associated with alterations of other established endpoints of toxicological concern. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Polychlorinated biphenyls (PCBs) are persistent organic contaminants that have been widely used as e.g. heat transfer fluids, lubricants, dielectric fluids in transformers and capacitors, flame retardants and sealants (Giesy and Kannan, 1998). PCBs show slow biodegradation and bioaccumulate in biota due to their chemical stability and lipophilic properties (Safe, 1994) and although their production and use have been banned in most countries for decades (Giesy and Kannan, 1998), PCBs are still present in closed sources, such as electrical transformers, and hence they continue to be released into the environment (Schmidt, 2010) and continue to contribute to human and wildlife exposure. PCBs induce a broad spectrum of biochemical and toxic effects in humans and animals (ATSDR, 2000). Fetuses and infants are considered particularly sensitive to chemical exposure due to their critical developmental periods (Makri et al., 2004). Studies in children have shown associations between PCB exposure and, e.g. neurotoxicity (Chen et al., 1992a; Grandjean and Landrigan, 2006), respiratory infections (Dallaire et al., 2006; Donaldson et al., 2010) and impaired fetal growth and development (Dallaire et al., 2013; Sagiv et al., 2007). Animal studies have confirmed that PCBs cause neurotoxicity, immunotoxicity and reproductive/developmental toxicity as well as a range of other effects (ATSDR, 2000). Individual PCB congeners are from a toxicological perspective classified as either dioxin-like (DL) or non-dioxin-like (NDL). The DL PCBs consist of 12 planar congeners that bind with high affinity to the aryl hydrocarbon receptor (AhR) and induce DL toxic effects. These congeners have been assigned toxic equivalency factors (TEFs) (Van den Berg et al., 2006) which can be used to estimate toxic equivalencies (TEQ) of combined exposures using the prototype dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as reference compound. The NDL PCBs consist of 197 non-planar congeners that bind with low affinity or not at all to the AhR and thus do not exhibit a typical DL toxicity pattern but may modulate other signaling pathways (Elabbas et al., 2013; Kretschmer and Baldwin, 2005). Several types of commercial PCB mixtures have been produced of which Aroclor 1254 (A1254) is one of the most common. A1254 contain about 54% chlorine by weight and consist of DL and NDL PCB congeners (Kodavanti et al., 2001). In animal studies A1254 have produced short- and long-term toxic effects, including neurotoxicity, immunotoxicity and developmental/reproductive toxicity (Bowers et al., 2004; Donaldson et al., 2010; Safe, 1990, 1994; Tilson and Kodavanti, 1998). However, few studies have investigated the toxicity of A1254 following perinatal exposure, in particular with regard to the retinoid (vitamin A) system. Morse and Brouwer (1995) observed reduced growth and changes in hepatic retinoid levels in rat offspring until postnatal day (PND) 90 and changes in organ weights until PND21 following gestational exposure to A1254. Chu et al. (2008) showed that A1254 induced growth suppression and changes in organ weights and biochemical parameters in rat offspring until PND77 following gestational and lactational exposure. One of the endpoints which showed a high effect size and was still affected at PND350 was decreased hepatic retinoid levels. The retinoid system, which is vital for developmental programming and morphogenesis, embryonic and postnatal growth,

immune function and reproduction (Blomhoff and Blomhoff, 2006) is under complex regulation by multiple enzymes, binding proteins and hormonal receptors as briefly indicated in Fig. 1. The diet-derived hormone precursors are stored in the liver and other tissues mainly as retinyl palmitate, and can, as needed, be converted 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-RA and its metabolite 9-cis-RA via the nuclear retinoic acid receptor (RAR) and retinoid X receptor (RXR) families (Balmer and Blomhoff, 2002). The all-trans-RA metabolite 9-cis-4-oxo-13,14dihydro-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 mixtures (Elabbas et al., 2014) and a marker of dietary vitamin A intake (Schmidt et al., 2003b). Rodent studies have shown that alteration of tissue retinoid levels, i.e. reduction of hepatic retinoids and increase of renal retinoids, are among the most sensitive endpoints following exposure to dioxins and DL PCBs (Chen et al., 1992b; Chu et al., 2001; Fletcher et al., 2001, 2005; Håkansson et al., 1991a,b; Van Birgelen et al., 1994a,b). Functional consequences of a modulated retinoid system during development are expected to be adverse and, hence, effects on the retinoid system has been considered an important endpoint and proposed for inclusion into testing guidelines for evaluating endocrine modulating properties of chemicals (OECD, 2012). The aim of the present study was to further clarify alterations of the retinoid system in rat offspring and their dams following gestational and lactational exposure to A1254, with a focus on individual retinoid forms including the signaling forms all-trans-RA and 9c-4o-13,14dh-RA, in liver, kidneys and serum. Also, differences in response between sex or between offspring and dams was evaluated and a partial least square (PLS) regression was performed to assess associations between alterations of tissue retinoid levels and already established biochemical and toxicological endpoints following A1254 exposure. It was also evaluated to which extent DL components of A1254 were causing the observed effects by using the 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 least HPLC 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 Canadian Council on Animal Care guidelines and approved by the Animal Care Committee of Health Canada. Details of animal housing, experimental design, gross and clinical observations 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 mg A1254/kg body weight (bw) from gestation day 1 until weaning on PND23. The dosing 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. Blood and tissues of dams were collected seven days after weaning, i.e. on PND30-32; while blood and tissues from one male and one female rat offspring from each litter were sampled at PNDs 35, 77 and 350, respectively. Blood and tissue materials were kept

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

43

* REOH-RBP-TTR (in plasma)

REOH

CRABP-RA

*

ADHs RDHs

REH

Retinyl esters

Retinoyl glucuronides

RALDHs, CYP 1A1, 1B1

RAL

*

UGTs

*

CYP26, CYP 2B, 3A

all-trans-RA

OH-/Oxo-RA

LRAT

*

CRBP-REOH

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

*

*

RXR NRs

RAR RXR

Development and growth Metabolism and homeostasis Reproduction and vision

Fig. 1. Retinoid metabolism, transport and gene regulation. Retinoic acid (RA) required for transcriptional activation of RAR-RXR heterodimers is usually produced from retinol (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 ensure proper 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 involve several 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, cytosolic alcohol dehydrogenase; RDH, retinol dehydrogenase; RALDH, retinal dehydrogenase; CYP, cytochrome P450 superfamily; CRABP, cellular retinoic acid binding protein; RAR, retinoic acid receptor; RXR, retinoid X receptor; NRs, nuclear receptors (e.g. thyroid hormone receptor); UGT, UDP-glucuronosyltransferase; *Indicates enzymes/proteins known to be affected by chemicals; →, biotransformation; −, binding equilibrium. Dashed lines indicate uncertainties in current knowledge. For overview references see Gomaa et al. (2012), Nilsson and Håkansson (2002), Novák et al. (2008), OECD (2012), Roos et al. (2011), and Zile (2001).

frozen at −80 ◦ C for subsequent analyses. Body and organ weights, serum thyroid hormone levels and hepatic cytochrome P450 (CYP) activity data (ethoxyresorufin O-deethylation (EROD), pentoxyresorufin O-dealkylation (PROD) and benzyloxyresorufin O-dealkylation (BROD)) are presented for all the time-points, i.e. PND35, 77 and 350 (see Section 2.4.1). PND35 data were previously presented in tabular format by Chu et al. (2008) while PND77 and 350 data were mentioned in text only when significances were observed. 2.3. Analyses of retinoids, CYP activities and thyroid hormones Retinyl palmitate and retinol in livers and kidneys were analyzed for both sexes at all time-points and dams by HPLC as described in Nilsson et al. (2000). Briefly, retinoids were extracted from tissues using diisopropyl ether and separated on a Nucleosil C18 5-␮m HPLC column (Macherey-Nagel, GmbH, Germany) using an ethanol:water (90:10, v/v) mobile phase and detected with a JASCO821-FP fluorescence detector (ex = 325 nm, em = 475 nm). Samples for retinyl palmitate and retinol in serum, that were only available for female offspring at PND77 and dams, were separated on a J’sphere ODS-H80 column (4.6 mm × 150 mm, 4-␮m particle size) obtained from YMC (Schermbeck, Germany) and detected with an ultraviolet detector at 325 nm as described by Schmidt et al. (2003b). The limit of detection (LOD) for retinol was 60 ng/g tissue or 1.2 ng/ml serum. The LOD for retinyl palmitate was 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 for both sexes at all time-points and for dams, and in serum for female offspring at PND77 and for dams by HPLC as previously described (Schmidt et al., 2003b). Briefly, separation of all-trans-RA and 9c-4o-13,14dh-RA from retinol and retinyl palmitate was achieved by solid-phase-extraction using an aminopropyl phase. Alltrans-RA and 9c-4o-13,14dh-RA were then separated on a Spherisorb ODS2 column (2.1 mm × 150 mm, 3 ␮m particle size) obtained from Waters (Eschborn, Germany) using a binary gradient, and were detected with a UV/vis detector at 340 nm. The metabolite, 9c-4o-13,14dh-RA, was extracted as described elsewhere (Schmidt et al., 2002). Since there is no available synthetic standard, levels of 9c-4o-13,14dh-RA were calculated using the calibration of 13-cis-4-OH-RA (Schmidt et al., 2002). The LOD for all-trans-RA was 0.2 ng/g tissue or 0.14 ng/ml serum. The LOD for 9c-4o13,14dh-RA was 0.3 ng/g tissue or 0.21 ng/ml serum. Hepatic CYP1A, CYP2B and CYP3A induction were assessed as EROD, PROD and BROD activities as previously described (Chu et al., 2008). Total serum thyroxine (T4 ) triiodothyronine (T3 ) and thyroid stimulating hormone (TSH) levels were analyzed using commercial radioimmunoassay kits as described in Chu et al. (2008) with LODs of 5.0 ng/ml, 0.2 ng/ml and 1.0 ng/ml, respectively. 2.4. Data evaluation 2.4.1. Statistical analysis Variables showed a normal distribution according to Kolmogorov–Smirnov nonparametric tests and Q–Q plots. Thus, data were described by mean ± standard

deviation (SD). Values for any sample that was below LOD were set to LOD/2. Differences between exposure groups of female and male offspring at PNDs 35, 77 and 350 as well as of dams at PND30 were tested against corresponding controls using two-tailed independent-samples T test. The level of significance was set at a pvalue of 0.6 was considered to be acceptable in order to study associations 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 entering significant variables in the PLS model (Chong and Jun, 2005). VIP values were computed as follows:

  h h   2 VIPj = p (SS(bk tk )(wjk /  wk ) / SS(bk tk ) k=1

k=1

SS(bk tk ) = b2k tkt tk Endpoints (X) and retinoid variables (Y) were visualized on correlation circles using their correlations with PLS components.

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

The PLS method was applied for offspring at PNDs 35, 77 and 350 as well as dams, which described the time-course effects of A1254 on hepatic retinoid levels by using two components. The applied PLS models explained 91% and 88% of the variance of endpoints (X) in female and male offspring at PND35, 87% of variance for dams, 73% and 62% of variance for female and male offspring at PND77, and 67% and 57% of variance for female and male offspring at PND350 (Supplementary Table 1). The applied PLS models explained 83% and 73% of the variance in retinoid concentrations (Y) of female and male offspring at PND35, 81% in dams, 60% and 51% in female and male offspring at PND77, and 59% and 58% of variance for female and male offspring at PND350 (Supplementary Table 1). The models with two components described A1254-related hepatic retinoid alterations in offspring at PND35 and dams better than they did in offspring at PNDs 77 and 350 (Supplementary Table 1). In fact, the models were optimized to fit A1254-related effects (Supplementary Table 1), which were apparent in offspring at PND35 and dams, while A1254 effects were subtle at PNDs 77 and 350. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.toxlet.2014.04.021.

2.4.3. Calculation of TCDD equivalents of A1254 To estimate the DL activity of A1254 changes in retinoid levels in this study as well as changes in body and organ weights and thyroid hormone levels were quantitatively compared to the effect of TCDD on these measures from a separate TCDD dose–response study using the same rat strain (Sprague Dawley) with a similar study design as the present study (Håkansson, H., personal communication; Finnilä et al., 2010; Giese et al., 2007) using the procedure previously described (Elabbas et al., 2011). Briefly, the TCDD equivalents were calculated as the dose which caused the same effect size (%) in the dose–response relationships of the TCDD study as in the present study at 15 mg A1254/kg bw. The TCDD equivalents of A1254 were compared to the theoretical daily TEQ of A1254, which was derived from the chemical composition of A1254 described in Elabbas et al. (2011). The TEQ was calculated to

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

3. Results 3.1. Hepatic retinoids Hepatic retinyl palmitate levels were decreased in female offspring at PNDs 35, 77 and 350 by 67%, 20% and 30%, respectively (Table 1). Similarly, hepatic retinyl palmitate levels were reduced in male offspring by 53%, 21% and 31% at PNDs 35, 77 and 350, respectively (Table 1). Hepatic retinol levels were reduced in female and male offspring at PND35, by 54% and 50%, respectively, but no significant changes were observed in offspring at PNDs 77 or 350 (Table 1). Hepatic all-trans-RA levels were increased at PND 35 in female and male offspring by 60% and 43%, respectively, but were not altered at PNDs 77 and 350 (Table 1). Hepatic 9c-4o-13,14dh-RA levels in female and male offspring at PND35 were reduced by 93% and 70%, respectively, though the reductions did not remain at 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 was decreased by 59% and its metabolite 9c-4o-13,14-dh-RA was below the LOD, thus reduced by >97% (Table 1).

Table 1 Concentrations of retinol, retinyl palmitate, all-trans-retinoic acid, and 9-cis-4-oxo-13,14-dihydro-retinoic acid (9c-4o-13,14-dh-retinoic acid) in livers and kidneys of Sprague Dawley offspring at postnatal days (PNDs) 35, 77 and 350 and dams at PND30 following maternal oral Aroclor 1254 from gestation day 1 until the end of lactation at 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 Offspring at PND77 Offspring at PND350 Dams at PND30

9–13 9–13 9–13 9–13

0.36 1.4 5.4 2.9

± ± ± ±

Hepatic retinol (nmol/g) Offspring at PND35 Offspring at PND77 Offspring at PND350 Dams at PND30

9–13 9–13 9–13 9–13

13 18 96 30

Hepatic all-trans-retinoic acid (pmol/g) 5 Offspring at PND35 Offspring at PND77 5 5 Offspring at PND350 5 Dams at PND30

Males 15 mg/kg

0 mg/kg

15 mg/kg

0.04 0.2 0.9 0.3

0.12 ± 0.02*** 1.1 ± 0.1** 3.8 ± 0.6*** 2.0 ± 0.3***

0.32 ± 0.04 1.0 ± 0.1 4.2 ± 0.6

0.15 ± 0.03*** 0.81 ± 0.08*** 2.9 ± 0.5***

± ± ± ±

3 4 66 4

6.2 ± 1.1 *** 15 ± 4 60 ± 19 20 ± 5***

12 ± 2 13 ± 3 59 ± 17

6.2 ± 1.1*** 12 ± 4 43 ± 15

15 23 26 41

± ± ± ±

1 11 8 8

24 ± 6* 15 ± 4 22 ± 14 17 ± 2**

14 ± 1 16 ± 4 35 ± 12

20 ± 5* 14 ± 6 51 ± 15

Hepatic 9c-4o-13,14dh-retinoic acid (pmol/g) 5 Offspring at PND35 Offspring at PND77 5 5 Offspring at PND350 5 Dams at PND30

15 30 46 35

± ± ± ±

5 7 12 5

1.0 ± 0.8*** 22 ± 7 47 ± 10