Toxicology Letters 184 (2009) 176–185

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Toxicology Letters

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

Contribution of methylmercury, polychlorinated biphenyls and organochlorinepesticides to the toxicity of a contaminant mixture based on Canadian Arcticpopulation blood profiles

Guillaume Pelletiera,∗, Sheila Massona, Mike J. Wadea, Jamie Nakaia, Ramona Alwisa,Susantha Mohottalageb, Premkumari Kumarathasanb, Paleah Blacka, Wayne J. Bowersa,Ih Chua, Renaud Vincenta

a Hazard Identification Division, Environmental Health, Science and Research Bureau, Chemicals Management Directorate, Health Canada, Canadab Mechanistic Studies Division, Environmental Health, Science and Research Bureau, Chemicals Management Directorate, Health Canada, Canada

a r t i c l e i n f o

Article history:Received 22 September 2008Received in revised form 7 November 2008Accepted 10 November 2008Available online 18 November 2008

Keywords:Environmental contaminantsMixtureRatNeurodevelopmentHypothyroidismTwo-dimensional protein electrophoresis

a b s t r a c t

Human populations are simultaneously exposed to a variety of anthropogenic contaminants. However,despite extensive literature on animal exposure to single compounds, data on the toxicity of complexmixtures are scarce. The Northern Contaminant Mixture (NCM) was formulated to contain the 27 mostabundant contaminants in the same relative proportions found in the blood of Canadian Arctic pop-ulations. Sprague–Dawley rat dams were dosed from the first day of gestation until weaning withmethylmercury (MeHg), polychlorinated biphenyls (PCBs) or organochlorines pesticides (OCs) admin-istered either separately or together in the NCM. An additional control group for hypothyroxinemiawas included by dosing dams with the goitrogen 6-propyl-2-thiouracil (PTU). Offspring growth, survival,serum thyroxine and Thyroid Stimulating Hormone (TSH) levels, thyroid gland morphology, brain taurinecontent and cerebellum and hippocampus protein expression patterns resulting from such exposureswere monitored. Pups’ increased mortality rate and impaired growth observed in the NCM treatmentgroup were attributed to MeHg, while decreased circulating thyroxine levels and perturbations of thy-roid gland morphology were mostly attributable to PCBs. Interestingly, despite comparable reduction inserum thyroxine levels, PCBs and PTU exposures produced markedly different effects on pup’s growth,serum TSH level and brain taurine content. Analysis of cerebellum and hippocampus protein expressionpatterns corroborated previous cerebellum gene expression data, as contaminant co-exposure in the NCM

significantly masked the effects of individual components on protein two-dimensional electrophoresispatterns. Identification by MALDI-TOF/TOF MS of differentially expressed proteins involved notably inneuronal and mitochondrial functions provided clues on the cellular and molecular processes affected by

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. Introduction

Long-range transport of persistent environmental contaminantsas led to significant accumulation of these toxicants in the Arc-ic environment and biota (Macdonal et al., 2000). Consumption of

sh and marine mammals is an integral part of the cultural identityf many Canadian Arctic communities. However, ingestion of thisraditional diet results in contaminant body burdens higher thanhose experienced by southern populations (Dewailly et al., 1994).

∗ Corresponding author at: Environmental Health Centre, 50 Colombine Drive-ay, P.L. 0803B, Tunney’s Pasture, Ottawa, Ontario, K1A 0L2 Canada.

el.: +1 613 941 8063; fax: +1 613 948 9413.E-mail address: guillaume pelletier@hc-sc.gc.ca (G. Pelletier).

378-4274/$ – see front matter. Crown Copyright © 2008 Published by Elsevier Ireland Ltoi:10.1016/j.toxlet.2008.11.004

n Copyright © 2008 Published by Elsevier Ireland Ltd. All rights reserved.

To investigate the possible health consequences of such exposure,the Northern Contaminant Mixture (NCM) was formulated to con-tain the 27 most abundant environmental contaminants at the sameproportion measured in the blood of Canadian Arctic Populations(Bowers et al., 2004; Chu et al., 2008).

In utero and lactational exposure of rats to the NCM has beenshown to affect pup growth, organ weight and histology, thyroidphysiology, liver enzymatic activities and blood biochemistry (Chuet al., 2008). In this follow-up study, the NCM was broken downinto its three main chemical classes: Rats were exposed perina-

tally to the NCM or to methylmercury (MeHg), polychlorinatedbiphenyls (PCBs) and organochlorine pesticides (OCs) administeredseparately at the same concentration found in the NCM, at twodose levels (see Table 1). Some of the contaminants were closeto, or above their LOAEL at the 100× dose level (Table 2), while

d. All rights reserved.

G. Pelletier et al. / Toxicology Letters 184 (2009) 176–185 177

Table 1Contaminant mixture treatment groups.

Treatment groups

Contaminants (mg/kg b.w./day) 100× NCM 100× PCBs 100× OCs 100× MeHg 1× NCM 1× PCBs 1× OCs 1× MeHg

PCBsa 1.1 1.1 0.011 0.011OC pesticidesa 1.9 1.9 0.019 0.019M

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he daily amounts of polychlorinated biphenyls, organochlorine pesticides and meta A complete list of PCB congeners and organochlorine pesticides is provided in S

he 1× NCM dose resulted in maternal blood contaminant levelshat approximated actual human blood levels in exposed Northernopulations (Chu et al., 2008). Due to the expected perturbationf thyroid hormone homeostasis by environmental contaminants,ne additional treatment group was exposed to the goitrogen 6-ropyl-2-thiouracil (PTU), to allow direct comparison of the effectsf environmental contaminants to those of mild hypothyroxine-ia.Data on pup’s tissue contaminant levels, histology, clinical bio-

hemistry and neurobehaviour resulting from exposure to thesenvironmental contaminant mixtures will be published elsewhere.nalysis of the effects of perinatal exposure to NCM, MeHg, PCBs,Cs and PTU on rat cerebellum gene expression at post-natal day 14as already been published (Padhi et al., 2008). This investigationevealed that co-exposure in the Northern Contaminant Mixturean significantly mask the effects of MeHg, PCBs and OCs admin-stered separately on brain gene expression. Despite this generalonclusion, the gene coding for cysteine-sulfinate decarboxylase,he rate limiting enzyme of taurine synthesis (Schuller-Levis andark, 2003), was found to be down-regulated across treatment

roups, genders (Padhi et al., 2008) and brain structures (unpub-ished data). Low plasma taurine level in infants has been correlatedo adverse effects on neurodevelopment (Wharton et al., 2004).n rats, hypothyroidism has been shown to decrease the serumoncentration of this semi-essential amino acid (Baskin et al.,

able 2he Northern Contaminant Mixture.

Dose (mg/kg) TEF LOAE

CB congeners28 0.007252 0.015499 0.0973101 0.0145105 0.0165 0.00003118 0.0727 0.00003128 0.0071138 0.2146153 0.3177156 0.0290 0.00003170 0.0562180 0.1522183 0.0193187 0.0795Sum PCBs 1.0992 0.25a

Sum TEF 3.55 × 10−6 1.0 ×rganochorine pesticidesAldrin 0.0049 0.025Dieldrin 0.0223 0.05b

�-HCH 0.0746 0.50a

Chlordane + metabol. 0.4319 0.75b

p,p′-DDT + p,p′-DDE 0.9756 0.25b

Hexachlorobenzene 0.2961 0.29b

Mirex 0.0291 0.70b

Toxaphene 0.0699 0.25a

Methylmercury chloride 1.997 0.125

mounts of each constituent provided in the 100× NCM dose are listed. Dioxin TEFs arummed with the parent compound, see Section 2 for a complete listing of all the componafety (www.inchem.org) and “b” from U.S. EPA Integrated Risk Information System (www

2.0 0.02 0.02

rcury chloride administered are listed for each treatment group.2.

1979), while increasing it in the developing brain (Heinonen,1975). These observations warranted further investigation onthe effects of environmental contaminants on brain taurine lev-els.

In the present report we will: (i) confirm and expand upon theobservations made by Chu et al. (2008), by determining the contri-bution of MeHg, PCBs, and OCs to the systemic toxicity of the NCM,(ii) compare the effects of perinatal exposure to environmental con-taminants with PTU-induced hypothyroxinemia, (iii) measure theeffects of exposure to environmental contaminants on brain taurinecontent, (iv) analyse cerebellum and hippocampus protein two-dimensional electrophoresis patterns to determine if the previouslydescribed loss of gene expression signature resulting from con-taminants co-exposure (Padhi et al., 2008) is also observed at theprotein expression level, and (v) identify differentially expressedproteins by MALDI-TOF/TOF MS to gain insights into the molecularmechanisms underlying neurotoxicity.

2. Materials and methods

2.1. Dosing mixtures

The formulation of the Northern Contaminant Mixture has been previ-ously described (Chu et al., 2008). Briefly, 1 ml of 100× NCM dissolved incorn oil (Mazola brand, Bestfoods, Canada) contained methylmercury chlo-ride (1.997 mg), organochlorine pesticides [p,p′-DDE (0.9187 mg), p,p′-DDT

L (mg/kg) Dose/LOAEL Comments

4.40 Aroclor 1254 exposure10−6a 3.55 TCDD exposure

b 0.200.450.150.58 Technical Chlordane mouse exposure3.90 Technical DDT exposure1.020.040.28 Camphechlor exposure

a 15.98 Multigenerational study

e provided for the relevant PCB congeners. Pesticide metabolites and isomers areents. “a” indicates LOAELs derived from the International Programme on Chemical.epa.gov/iris).

1 gy Letters 184 (2009) 176–185

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Table 3Pups mortality rates.

Treatment group Pups mortality PND 1–4 Pups mortality PND 5–21

Control 3.1% (3/13) 0% (0/13)PTU 1.2% (2/11) 1.6% (2/11)1× NCM 0.7% (1/11) 0% (0/11)1× MeHg 0% (0/6) 0% (0/6)1× PCBs 1.4% (2/11) 0.7% (1/11)1× OCs 2.9% (2/8) 0% (0/8)100× NCM 33.1%* (13/13) 4.1% (1/10)100× MeHg 46.7%* (11/11) 5.8% (4/8)100× PCBs 2.4% (1/10) 0.8% (1/10)100× OCs 2.9% (4/9) 0% (0/9)

Mortality is expressed as the percentage of pups that perished and the proportion of

(Bio-Rad) according to the manufacturer’s protocols. Stained gels were scannedon a GS-800 Calibrated Densitometer (Bio-Rad) and protein spots were detected,matched, quantified and normalized (% total spot volume) using Phoretix 2D soft-ware (Nonlinear USA, Durham, NC, USA). Normalized spot intensity ratios from 7to 10 samples per treatment group were log-transformed, exported to Genespring

78 G. Pelletier et al. / Toxicolo

0.0569 mg), hexachlorobenzene (0.2961 mg), trans-nonachlor (0.2203 mg), oxy-hlordane (0.1359 mg), �-heptachlor epoxide (0.0232 mg), aldrin (0.0049 mg), �-exachlorocyclohexane (0.0746 mg), cis-nonachlor (0.0525 mg), mirex (0.0291 mg),ieldrin (0.0223 mg) and toxaphene (0.0699 mg)] and PCBs [PCB congeners 280.0072 mg), 52 (0.0154 mg), 99 (0.0973 mg), 101 (0.0145 mg), 105 (0.0165 mg), 1180.0727 mg), 128 (0.0071 mg), 138 (0.2146 mg), 153 (0.3177 mg), 156 (0.029 mg), 1700.0562 mg), 180 (0.1522 mg), 183 (0.0193 mg) and 187 (0.0795 mg)]. PTU was dis-olved at 10 ppm in drinking water.

.2. Animal treatment and tissue collection

Sprague–Dawley rats were purchased from Charles River Laboratories (St-onstant, QC, Canada) and kept in polycarbonate hanging cages on shaved woodedding. The room was kept at 22 ± 2◦C and 50 ± 10% humidity, on a 12 h light cycle.ood (Purina Mills Lab Diet 5001) and water were provided ad libitum. All experi-ental procedures adhered to the Canadian Council on Animal Care guidelines andere approved by Health Canada’s Institutional Animal Care Committee.

Female rats (250–275 g) were allowed to acclimatize to the animal facility for0 days and trained to accept a sweet cookie (Teddy Graham, Kraft Canada, Canada).ifteen females per treatment group were bred in order to obtain 10 litters. Twoemales were introduced in the cage of a male rat and monitored twice-daily forhe presence of a vaginal plug. Upon detection of copulation, females were removedrom the male’s cage to be singly housed and randomly assigned to a treatmentroup. Dams were dosed from gestation day 1 (GD 1) to post-natal day 21 (PND 21)ith a cookie plus a weight-adjusted volume of corn oil (1.0 ml/kg b.w.) laced with

he appropriate concentration of contaminants. All rats consumed their cookie iness than 5 min. The Northern Contaminant Mixture, methylmercury, organochlorineesticides and polychlorinated biphenyls were administered at two dose levels (1×nd 100×, see Table 1). The control group for hypothyroxinemia was generated byosing dams with 10 ppm PTU in drinking water from GD 6 to PND 21. Dams fromhe control and PTU treatment groups also received a cookie with a weight-adjustedolume of corn oil from GD 1 to PND 21. Females were monitored daily and the dayf birth (PND 0) recorded. Offspring were counted and weighed at birth and culledo 4 males and 4 females per litter on PND 4. Mortality and morbidity were recorded.ams and pups were weighed daily.

On PND 14 and 21 two pups per sex per litter were sacrificed. For brain pro-eomic analysis, one male and one female pup were decapitated without anesthesiand their brains quickly extracted, rinsed with ice-cold PBS, sliced in half alonghe longitudinal fissure, frozen on crushed dry ice and kept at −80◦C. The secondair of pups were exsanguinated via cardiac puncture under isoflurane anesthesia,he blood collected in SST Vacutainer tubes (BD Canada, Oakville, ON, Canada), anderum stored at −80◦C. The trachea was dissected clear of connective tissue andmmersed in PBS + 4% paraformaldehyde for analysis of thyroid gland morphology.

.3. Thyroid hormone and gland morphology measurements

Serum thyroxine (T4) and Thyroid Stimulating Hormone (TSH) were measuredsing commercially available RIA kits (Cat. No. 06-B254030, ICN Biomedicals, Aurora,H, USA and Cat. No. RPA554, GE Healthcare, Piscataway, NJ, USA). Thyroid glandorphology was assessed as previously described (Wade et al., 2002). Briefly,

araffin-embedded samples were cut into 5 �m thick slices along the trachea axisnd stained with Periodic Acid Schiff. Two 625 × 410 �m images from 2 separate his-ological sections from 5 animals per treatment group were acquired in Image-Prolus software (Media Cybernetics, Silver Spring, MD, USA). Epithelial cell thicknessas quantified by one technician unaware of the sample treatment.

.4. Taurine measurement

Brain tissue was homogenized in 5 volumes of 0.2 M perchloric acid using anltrasonic tissue homogenizer and then centrifuged for 20 min at 10,000 × g atoom temperature. The protein content of the pellets was measured using a mod-fied Lowry assay (Cat. No. TP0200, Sigma–Aldrich Canada, Oakville, ON, Canada)nd supernatant taurine measurement was based on the method of McCarthy et al.2000). Briefly, 50 �l of the supernatant or taurine standard solution (0–400 �g/ml)as mixed with 250 �l of 0.2 M Na2CO3 (pH 9.7), followed by addition of 250 �lf dansyl chloride solution (10 mg/ml in 70% acetonitrile [ACN]) and further mix-ng. The samples were incubated for 30 min at 65◦C, during which they were mixedgain half way through the incubation. After cooling to room temperature, 50 �l of% H3PO4 was added and the samples vortexed before addition of 250 �l 0.5 M phos-hate buffer (pH 6.2) and 150 �l water. The reaction mixtures were spun for 6 mint 8000 × g and the supernatants collected into amber autosampler vials for HPLCnalysis (Agilent 1100 Series HPLC System and 1200 Series Fluorescence Detector).amples (20 �l) were injected onto a Supelco Supelcosil LC-18-DB column (5 �m,

5 cm × 4.6 mm) held at 35◦C. The dansylated taurine derivative was eluted at a flowate of 1.5 ml/min using a linear gradient of 12–17.5% ACN in 0.02 M phosphate bufferpH 3.0) over 21 min and detected by fluorescence (220 nm excitation wavelength,30 nm emission). The column was then washed with 48% ACN for 3 min followedy 5 min equilibration back to the starting conditions prior to the subsequent samplenjection.

litters affected by mortality is indicated between brackets. PND 1–4 reports deathsthat occurred before PND 4 culling and PND 5–21 from culling to the termination ofthe study. *Significantly different from control group values.

2.5. Proteomic analysis

2.5.1. Two-dimensional electrophoresisTwenty volumes of extraction buffer (8 M urea, 50 mM DTT, 2% (w/v) CHAPS

and 0.2% (w/v) BioLyte 5/8 Ampholyte (Bio-Rad Laboratories, Hercules, CA, USA))were added to dissected cerebellum or hippocampus before sonication on ice forone minute. Extracts were then centrifuged for 15 min at 16,000 × g at room tem-perature and the supernatants collected, aliquoted and stored at −80◦C. Bio-RadReadyStrip IPG strips (pH 5–8, 7 cm) were rehydrated overnight at room temperaturewith 125 �l of protein extract, containing approximately 300 �g of protein. Electro-focussing (12,000 V/h, 50 �A current limit per strip and linear voltage gradient up to4000 V) was performed on a Protean IEF Cell (Bio-Rad). Strips were then equilibratedfor 10 min in 2.5 ml of equilibration buffer (6 M urea, 2% (w/v) SDS, 0.375 M Tris–HClpH 8.8, 20% (v/v) glycerol) containing 2% (w/v) DTT and for another 10 min in equili-bration buffer containing 2.5% (w/v) iodoacetamide. Strips were then placed on topof 12% polyacrylamide gels, electrophoresed and stained with Bio-Safe Coomassie

Fig. 1. Body weights of (A) dams and (B) pups (male and female average) followingexposure to 100× NCM, 100× MeHg, 100× PCBs, 100× OCs and PTU. Error barsrepresent S.E.M.

G. Pelletier et al. / Toxicology Letters 184 (2009) 176–185 179

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ig. 2. Effects of contaminant and PTU exposures on the thyroid hormone system. (AND 21. (D) Thyroid epithelial thickness at PND 21. Error bars represent standard de

X 7.3 (Agilent Technologies, Santa Clara, CA, USA), where treatment effects weressessed by ANOVA, followed by Benjamini and Hochberg False Discovery Rate forultiple testing correction and Student-Newman-Keuls post hoc test, with p < 0.05

s a cut-off value for statistical significance.

.5.2. MALDI-TOF/TOF MS analysisProtein extracts were pre-fractionated on a MicroRotofor (Bio-Rad) according to

he manufacturer’s instructions. Differentially expressed protein spots were excisedrom several preparative 2D gels and stored at −80◦C until MALDI-TOF/TOF MS asescribed by Kumarathasan et al. (2005). Briefly, gel cuts were dehydrated withCN and washed with 50 mM NH4OAC in 30% ACN for destaining. Salts and SDSere removed by sequential incubation with methanol, chloroform and water,

espectively. Dried gel plugs were digested overnight with trypsin solution at 37EC.upernatants were collected after centrifugation (8500 × g, 20 s.) and sequentiallyxtracted with 0.1% TFA (aq), 30% ACN (0.1% TFA) and 60% ACN (0.1% TFA). Allupernatants were combined, dried and reconstituted in 30% ACN (0.1% TFA). Theatrix used for mass spectrometric analysis was �-cyano-4-hydroxycinnamic acid

Bruker Daltonics, Billerica, MA, USA) in 50% ACN (0.1% TFA). Sample and matrixere spotted on an AnchorChip target plate and analyzed by Autoflex MALDI-

OF/TOF MS (Bruker Daltonics) employing the experimental conditions reportedarlier (Kumarathasan et al., 2005). Mass spectral data were matched againstwiss-Prot, NCBInr, and MSDB for PMF, using MASCOT search engine (Matrix Sci-nces).

.6. Western immunoblotting

Following two-dimensional electrophoresis, gels were transferred to Hybond-

Extra membranes (GE Healthcare) and proteins visualized with SYPRO Ruby

rotein blot stain (Bio-Rad) according to the manufacturer’s protocol. HSP60 andubulin beta rabbit polyclonal antibodies were purchased from Cell Signaling Tech-ology (New England Biolabs, Pickering, ON, Canada) and immunoblotting waserformed as suggested by the manufacturer, using horseradish peroxidase con-

ugated secondary antibody and ECL Plus Western Blotting Detection System (GEealthcare).

m total T4 concentration at PND 14 and (B) PND 21. (C) Serum TSH concentration atns and treatment groups identified by the same letter are not statistically different.

2.7. Statistical analyses

Statistical analyses were performed using SPSS 15.0 software (SPSS Inc., Chicago,IL, USA). Shapiro-Wilk and Levene tests were used to assess data normality andhomogeneity of variance. Data were analyzed by ANOVA and specific treatmenteffects identified by Tukey’s post hoc test. Mortality rates and other datasets whichdid not satisfy ANOVA assumptions were assessed using Kruskal-Wallis ANOVAfollowed by Mann–Whitney U-test, if required. Dam and pup daily weights wereaveraged in blocks of 3 days or 4 days and the repeated measures analyzed using GLMfollowed by Tukey’s post hoc test. Treatment effects were considered statisticallysignificant for p < 0.05.

3. Results

Exposure to the 1× Northern Contaminant Mixture (0.05 mgNCM/kg b.w./day) has been shown to result in rat maternal bloodcontaminant concentrations at PND 30 that approximate actualhuman blood profile (Chu et al., 2008). In the present study, the1× NCM (0.05 mg/kg b.w./day), 1× MeHg (0.02 mg/kg b.w./day),1× PCBs (0.011 mg/kg b.w./day) and 1× OCs (0.019 mg/kg b.w./day)treatment groups did not result in any significant effects on pupssurvival (Table 3), growth (Fig. 1B) and thyroid hormone levels(Fig. 2A–C).

3.1. Growth and mortality

Litter size and gender ratio were not affected in any treatmentgroup (data not shown). Statistically significant effects on pup sur-vival were observed in the 100× NCM and 100× MeHg treatmentgroups (Table 3). Mortality was observed in all litters of these treat-ment groups. Three litters in both treatment groups completely

1 gy Letters 184 (2009) 176–185

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ied before PND 4, resulting in 33% and 46% mortality rates, respec-ively. After PND 4, the 4.1% mortality rate resulting from exposureo 100× NCM was attributable to high mortality in one litter, whilehe 5.8% mortality rate observed in 100× MeHg involved four differ-nt litters. However, the seemingly higher mortality rates observedn the 100× MeHg treatment group were not statistically differentrom those observed in the 100× NCM.

None of the treatments significantly affected dam weightsefore the end of gestation (Fig. 1A). After birth, the only statis-ically significant difference in dam weights was observed betweenhe PTU and 100× MeHg treatment groups. In pups, growthurves resulting from 100× NCM, 100× MeHg and PTU expo-ures were significantly different from the control group (Fig. 1B).TU treatment resulted in a growth curve that was relativelylose to control and 100× PCBs values for the first two post-atal weeks, but then reached a plateau to finally approximate00× MeHg values by PND 21. The 100× NCM growth curve,ying halfway between control and 100× MeHg values was alsoignificantly different from the 100× MeHg growth curve. How-ver, this latter observation has not been replicated in anotherohort of similarly treated rats (W.J. Bowers, unpublished obser-ation).

.2. Thyroid hormone homeostasis and thyroid gland morphology

No statistically significant gender differences or gender-reatment interactions were observed for thyroid parameters.onsequently data presented in Fig. 2 are the averaged values ofale and female pups. Effects of 100× PCBs and 100× NCM on

erum T4 and TSH and thyroid histology are very similar sug-esting that most of the effects of the NCM are attributable tohe PCBs. In rats, PND 14 corresponds to a peak in serum thy-oid hormone levels (Zoeller et al., 2007). The impaired growthbserved in 100× NCM, 100× MeHg and PTU treatment groupsFig. 1B) may also have delayed the appearance of this thy-oid hormone peak, resulting in an apparent exacerbation of theffect of these treatment groups at PND 14. This hypothesis isest exemplified by 100× MeHg which significantly lowered andhen increased serum T4 levels at PND 14 and 21 (Fig. 2A and), without affecting serum TSH and thyroid epithelial thick-ess (Fig. 2C and 2D). Exposures to PTU, 100× NCM and 100×CBs resulted in similar serum T4 levels at PND 21 (Fig. 2B)hich did not translate into a similar TSH response: PTU trig-

ered a 400% increase in serum TSH concentration, compared to50% increase for 100× NCM and 100× PCBs treatment groups

Fig. 2C).As expected, PTU, 100× NCM and 100× PCBs treatment groups

hich affected serum T4 and TSH levels also increased thy-oid epithelial cell thickness (Fig. 2D). In absence of measurableffects on serum T4 and TSH levels, exposure to 100× OCs nev-rtheless caused a mild but statistically significant thickeningf the thyroid epithelial layer. The induction of liver metabo-izing enzymes by organochlorine pesticides may also increase4 clearance, triggering a compensatory increase in T4 synthe-is and affecting thyroid gland morphology (Qatanani et al.,005).

.3. Brain taurine measurements

PND 14 cerebra remaining after hippocampus dissection were

sed for taurine measurement. PTU-induced hypothyroxinemiaas found to significantly increase brain taurine content (Fig. 3).owever, despite similar inhibition of serum T4 levels, 100× NCMnd 100× PCBs treatment groups had no effect on taurine concen-ration.

Fig. 3. Taurine levels in PND 14 male and female cerebra following exposure to 100×NCM, 100× MeHg, 100× PCBs, 100× OCs and PTU. Error bars represent standarddeviations. *Significantly different from control group values.

3.4. Analysis of two-dimensional protein electrophoresis patterns

Differentially expressed protein spots identified in male PND14 cerebellum and hippocampus are listed in Table 4A and B andtheir localizations on a two-dimensional gel presented in Fig. 4Aand B, respectively. A total of 19 spots in cerebellum and 16 spotsin hippocampus were found to be differentially expressed follow-ing exposure to 100× NCM, 100× MeHg, 100× PCBs or 100× OCs.Comparison of the differentially expressed spots in cerebellum andhippocampus reveals that only 3 spots were significantly affectedin both brain structures. Similar findings have been reported atthe gene expression level following perinatal exposure to PCBs(Royland and Kodavanti, 2008).

Despite the small overlap between the differentially expressedprotein spots in cerebellum and hippocampus, the same overallpattern emerged in both structures, i.e. the effects of the NorthernContaminant Mixture on protein expression patterns are markedlydifferent from those of MeHg, PCBs and OCs administered sepa-rately. In the cerebellum, out of the 17 differentially expressed spotsobserved in the 100× MeHg, 100× PCBs or 100× OCs treatmentgroups, only 3 were similarly affected in 100× NCM (Table 4A). Inthe hippocampus, out of 10 differentially expressed spots in 100×MeHg, 100× PCBs and 100× OCs treatment groups, only one (spot222) was significantly affected in the 100× NCM treatment group(Table 4B). A few protein spots (2 in the cerebellum and 6 in thehippocampus), while not significantly different from control groupvalues in 100× MeHg, 100× PCBs and 100× OCs treatment groupswere nevertheless found to be differentially expressed in the 100×NCM treatment group.

Analysis of protein expression patterns also provided insightsinto the perturbation of thyroid hormone-driven processes byorganochlorine compounds. In the cerebellum, about half of theproteins differentially expressed in 100× PCBs and 100× OCstreatment groups were similarly affected following PTU exposure(Table 4A). Similarities between 100× OCs and PTU treatmentgroups suggest that despite apparently normal circulating T4 levels(Fig. 2A and B) 100× OCs may affect thyroid hormone-dependantpathways in the brain, or alternatively that organochlorine pesti-cides and PTU can affect the same thyroid hormone-independentpathways (Etienne et al., 2003). However, the situation was dif-ferent in the hippocampus, where only one protein (spot 311)

was similarly affected in 100× PCBs, 100× OCs and PTU, andanother (spot 417) up-regulated in 100× PCBs and down-regulatedin PTU treatment group (Table 4B). These observations suggest bothhypothyroxinemia-dependant and independent effects of PCBs onbrain development.

G. Pelletier et al. / Toxicology Letters 184 (2009) 176–185 181

Fig. 4. Differentially expressed proteins in PND 14 male (A) cerebellum and (B) hippocampus following exposure to 100× NCM, 100× MeHg, 100× PCBs, and 100× OCs. Proteinspots presenting intensities significantly different from control group values in at least one treatment group are circled and numbered on control cerebellum and hippocampusprotein two-dimensional electrophoresis patterns. (C) Immunological confirmation of MALDI-TOF/TOF MS identification of HSP60 and Tubulin beta in hippocampus proteinsample.

Table 4Differentially expressed protein spots following exposure to 100× NCM, 100× MeHg, 100× PCBs and 100× OCs in PND 14 male (A) cerebellum and (B) hippocampus.

Spot 100× NCM 100× MeHg 100× PCBs 100× OCs PTU Protein Identity Access. number MOWSE score Sequence % cover.

(A)3 2.56 1.39 1.94 2.20 3.08 Albumin P02770 58 24

192 1.28 1.04 −1.08 8.99 6.41262 −1.55 1.04 −1.12 1.73 −1.17 NECAP-1 P69682 50 26282 1.34 −1.38 1.83 3.37 1.84292 −2.21 −1.88 −2.90 1.20 −3.31312 −2.06 −1.50 −4.38 −1.91 1.27338 1.38 −1.36 1.12 2.21 1.36346 1.06 −1.34 −1.02 1.35 1.21 MPST P97532 52 26350 −7.01 −1.16 1.30 −2.24 −1.23375 −6.04 1.17 1.02 −1.07 1.31 VDAC-2 P81155 52 26419 −3.75 n.d. −4.09 −1.31 n.d.429 1.17 −1.02 1.21 2.29 1.72 Ring finger

protein 141Q6IV57 69 31

444 1.31 −1.00 1.08 2.08 1.66466 1.58 −1.71 −2.56 6.72 −2.31478 1.46 −1.95 −1.17 2.28 1.21503 −2.07 −4.03 −1.88 −2.94 −6.16506 1.09 1.07 1.36 1.11 −1.05 HCNP precursor P31044 72 33563 n.d. −3.24 −2.63 1.11 −4.19565 −1.32 −1.10 −2.66 −1.30 −1.04

Spot 100× NCM 100× MeHg 100× PCBs 100× OCs PTU Protein Identity Access. number MOWSE score Sequence % cover. Immuno. confirm.

(B)29 2.80 3.07 3.39 4.77 1.44 Albumin P02770 67 20155 −1.35 1.17 1.28 1.19 2.36 HSP60 mito. prec. P63039 77 27 X159 −2.64 −1.04 1.06 1.27 1.72221 −1.09 1.09 1.21 1.27 −1.10 Tubulin beta-2B Q3KRE8 72 31 X222 1.48 1.23 1.67 1.53 1.41234 −1.07 1.14 1.22 1.09 1.34 NECAP-1 P69682 67 29262 −1.66 1.12 1.27 1.14 −5.98 NECAP-1 P69682 50 26306 1.01 1.65 1.33 1.30 1.65308 −1.21 −1.06 −1.65 −1.71 1.15 Tubulin beta-2B Q3KRE8 58 26 X311 1.78 −2.80 4.79 5.52 6.58338 1.25 −2.72 1.48 1.33 1.48395 −2.76 −1.11 1.04 1.08 −5.48417 −2.35 −1.66 2.41 1.19 −4.52448 −2.60 1.08 1.08 −1.69 −1.06563 −3.40 −1.16 1.04 −1.76 −3.06576 −1.15 1.91 1.27 −1.19 1.37

Expression fold changes relative to the control group are provided and statistically significant up or down-regulations are indicated in bold. NCBI accession number, MOWSEscore and protein coverage (%) are provided for proteins conclusively identified by MALDI-TOF/TOF MS. Proteins whose identity was further confirmed by subsequentimmunoblotting are identified by an “X” in the immunological confirmation column. n.d.—not detected.

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.5. Identification of differentially expressed proteins

MALDI-TOF/TOF MS analysis conclusively identified about ahird of the differentially expressed proteins following exposureo environmental contaminants (Table 4A and B). The expressionnd function in the brain of most of these proteins have been char-cterized. A large proportion of the identified proteins are linkedo mitochondrial functions (HSP60, VDAC-2 and MPST) or directlynvolved in neuronal functions (HCNP precursor, NECAP-1), pro-iding clues on molecular mechanisms of neurotoxicity that wille presented in the discussion.

Two antibodies were used to confirm MALDI-TOF/TOF MSdentification of differentially expressed proteins in hippocampusxtracts. Immunological signal of anti-Tubulin beta antibody co-ocalized with protein spots 221 and 308 identified as Tubulineta-2B by MALDI-TOF/TOF MS. However, neither of those 2 spotsepresented the bulk of Tubulin beta immunological signal (Fig. 4C,pper panels). Interestingly, up-regulation of spot 221 was accom-anied by a down-regulation of spot 308 in 100× PCBs and 100×Cs treatment groups, suggesting that these observations may beorrelated and correspond to a shift in post-translational modifica-ion state of a subpopulation of Tubulin beta. Similar results werebtained with anti-HSP60 antibody, as the bulk of the immunolog-cal signal located close to HSP60 calculated pI of 5.9 and molecular

eight of 60 kDa, while two weaker signals, collocated with spot55, identified as HSP60 by MALDI-TOF/TOF MS and spot 159 whichas not conclusively identified (Fig. 4C, lower panels). Interest-

ngly, spots 155 and 159 also appeared to be coordinately regulatedTable 4B).

The immunological confirmation of MALDI-TOF/TOF MS datauggests that two-dimensional protein electrophoresis patternnalysis identified changes in post-translational modification statesather than changes in Tubulin beta and HSP60 total protein levels.

. Discussion

Consumption of country food by Canadian Arctic populationsesults in environmental contaminant body burdens significantlyigher than those experienced by southern populations (Dewaillyt al., 1994) and epidemiological studies suggest that these contam-nants may adversely affect neurodevelopment (Van Oostdam et al.,005). In order to refine risk assessment, the Northern Contaminantixture was developed to reproduce the blood contaminant pro-

le from women of child bearing-age in the North-Eastern ArcticBowers et al., 2004). Contaminants in the 1× NCM, 1× MeHg, 1×CBs, and 1× OCs treatment groups were well below their respec-ive LOAELs (Table 2). Rat exposure to 1× NCM, which yields a serumontaminant profile in the dams that approximates actual humanxposure levels (Chu et al., 2008), did not produce any measurableffects on pups survival, growth or thyroid hormone homeostasis.or this complex combination of substances, the lack of observedffects in developmentally exposed animals suggests that there waso major synergy resulting in toxicity.

Hexachlorobenzene was administered approximately at LOAEL,CBs and p,p′ DDT + DDE at about 4 times their respective LOAELsnd MeHg at 16 times LOAEL in the 100× treatment groups (Table 2).or most of these compounds, hepatic lesions or enzymatic induc-ion in mature animals were reported and the use of variousxposure and measurement protocols may explain the large varia-ions in NOAELs and LOAELs reported in the literature. In addition,

echnical mixtures of PCBs and pesticides were used to determineOAELs, which may not adequately represent the PCB congeneratio or pesticide isoforms and/or metabolites found in biologicalamples. Given that levels of exposure for many of the individualomponents of the mixture exceeded the doses previously shown

tters 184 (2009) 176–185

to induce toxicity in similar animal models, observable toxicity wasexpected in the 100× dose treatment groups.

Table 3 shows that the only treatment groups clearly affectingthe survival of pups were the 100× NCM and 100× MeHg. Despitethe fact that mortality rates observed in 100× MeHg were about40% higher than those observed in 100× NCM before and afterculling, these two treatment groups were not statistically different.In this specific study, co-exposure with PCBs and OCs significantlymitigated the effects of MeHg on pup growth (Fig. 1B). How-ever, this interaction has not been consistently observed in othercohorts and should therefore be considered with caution. Inter-actions between methylmercury and organochlorine compoundshave been previously described. While a few studies on MeHg andPCBs co-exposure reported exacerbation of toxic effects (Bemis andSeegal, 1999, 2000; Fischer et al., 2008; Roegge et al., 2004), mostpublications on this topic reported either additive or antagonisticeffects (Coccini et al., 2006, 2007; Costa et al., 2007; Johansson etal., 2006; Sugawara et al., 2008; Vettori et al., 2006; Widholm et al.,2004).

Several differences between the effects of PCBs and PTU-inducedhypothyroxinemia were observed. Despite very similar serum T4levels at PND 21, PTU triggered a four-fold increase in serum TSHlevel while 100× PCBs and 100× NCM effects, although statisticallysignificant, remained comparatively modest (Fig. 2B and C). Thefailure of PCB-induced hypothyroxinemia to trigger an appropriateTSH response has been previously described (Gauger et al., 2007;Goldey et al., 1995; Khan and Hansen, 2003; Morse et al., 1996b). Aclear difference between pup growth curves resulting from expo-sure to 100× PCBs and PTU was also observed (Fig. 1B), indicatingthat perturbation of thyroid functions by PCBs are not sufficientto account for the growth retardation observed in the 100× NCMtreatment group. PTU-induced hypothyroxinemia resulted in sig-nificantly higher taurine concentrations in cerebrum, in agreementwith observations made on hypothyroid rats (Heinonen, 1975),while 100× NCM and 100× PCBs treatment groups had no effect onthis endpoint (Fig. 3). Finally, while a significant proportion of theeffects of 100× PCBs on cerebellum protein expression pattern mayhave been attributed to lower circulating thyroid hormone levels,100× PCBs and PTU effects on hippocampus protein pattern werevery poorly correlated, and even contradictory for one differentiallyexpressed protein. Divergent effects of PCB exposure and hypothy-roidism on other endpoints have also been reported (Roegge et al.,2006; Royland and Kodavanti, 2008). Taken together, these datasuggest that the well documented adverse effects of PCBs on neu-rodevelopment may be consequent to mechanism(s) of toxicity thatare at least partly independent of PCB-induced hypothyroxinemia.

Data on global gene expression resulting from exposure totoxicants administered either alone or as part of a mixture arescarce. However, it has been reported that toxicant co-exposurecan significantly mask the effects resulting from exposure to theindividual components administered separately (Finne et al., 2007;Hendriksen et al., 2007; Padhi et al., 2008). Analysis of brain pro-tein two-dimensional electrophoresis patterns presented in thisreport reinforces the conclusions drawn from genomic investiga-tions. Comparison of the differentially expressed proteins in bothcerebellum and hippocampus reveals that only a minority of theproteins identified in the 100× MeHg, 100× PCBs and 100× OCstreatment groups were similarly affected in the 100× NCM treat-ment group (Table 4A and B). This observation has significantimplications for risk assessment, as it suggests that biomarkers ofexposure/effects developed in single-compound exposure studies

may not be reliable in the context of exposure to complex mixtures.

Given the weak similarities between cerebellum and hippocam-pus responses to environmental contaminants and the fact thatmost of the proteins differentially expressed in the 100× MeHg,100× PCBs or 100× OCs treatment groups were not significantly

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ffected in the 100× NCM treatment group, the few proteins dif-erentially expressed in sub-mixtures and NCM and in both braintructures represent potentially interesting biomarkers of expo-ure/effects. Two such proteins were identified by MALDI-TOF/TOFS: Albumin (spot 3 in the cerebellum and 29 in the hippocampus)

nd Adaptin Ear-Binding Clathrin-Associated Protein 1 (NECAP-1,pots 234 and 262), see Table 4 and Fig. 4.

Albumin extravasation has been reported following exposure toeHg (Bertossi et al., 2004), while PCBs (Slim et al., 2001; Toborek

t al., 1995) and organochlorine pesticides (Sinha and Shukla, 2003)ave been shown to induce blood-brain barrier dysfunction in vivor in vitro. Therefore, the increased amounts of albumin detected inerebellum and hippocampus extracts following exposure to envi-onmental contaminant mixtures may be indicative of perturbationf cerebrovascular permeability. Immunostaining of brain histolog-cal sections will be required to confirm this hypothesis.

NECAP-1 is a clathrin accessory protein highly expressed inerebellum and hippocampus, which co-localizes in synapticesicles with synaptophysin and synaptotagmin (Murshid et al.,006). Interestingly, polychlorinated biphenyls exposure has beenhown to affect synaptophysin expression (Goodwill et al., 2007;alkiewicz et al., 2005; Morse et al., 1996a). The identification of

wo post-translational modification forms of NECAP-1 may providedditional information about the molecular mechanisms involvedn the perturbation of synaptic function by environmental contam-nants.

Out of the 8 differentially expressed proteins we were ableo conclusively identify in the cerebellum and/or hippocampus,

proteins, namely 60 kDa Heat Shock Protein mitochondrial pre-ursor (HSP60), 3-mercaptopyruvate sulfurtransferase (MPST) andoltage-Dependant Anion-selective Channel protein 2 (VDAC-2)re related to mitochondrial functions. This finding may be due tobias of protein 2D electrophoresis toward abundant mitochon-

rial proteins, but it is nevertheless interesting in light of the rolef mitochondria in cellular fate and neurodegeneration (Norenbergnd Rao, 2007). HSP60 participates in the folding of mitochondrialroteins and is generally considered as a marker of mitochondrialontent. However, it may also play a neuroprotective role by inter-cting with Bax and Bcl to affect the apoptotic cascade (Chan etl., 2007). Similarly, VDAC-2, a mitochondrial ion channel proteins also involved in dopamine-induced apoptosis (Premkumar andimantov, 2002) probably through interaction with Bax/Bak pro-eins (Chandra et al., 2005; Shoshan-Barmatz et al., 2006). MPSTs related to mitochondrial rhodanese but is found in both mito-hondria and cytoplasm (Nagahara et al., 1998), where its role inhe pyruvate synthesis pathway is essential for neuronal functionsNagahara and Sawada, 2006). Interestingly, HSP60 and VDAC-2own-regulation was observed only in the complete Northern Con-aminant Mixture treatment group.

Spot 506 was identified as Hippocampal Cholinergic Neurostim-lating Peptide (HCNP) precursor. This abundant, multifunctionalrotein has been shown to bind to phosphatidylethanolamine, opi-ids and ATP (Ojika et al., 2000) and to inhibit proteosome activityVan der Staay, 2006) and ERK phosphorylation (Matsukawa etl., 2003). Following stimulation of the NMDA receptors, prote-litic cleavage of this protein releases the 11 peptide-long HCNPOjika et al., 2000) which has been shown to stimulate choliner-ic activity (Taiji et al., 1996). The up-regulation of HCNP precursory PCBs may help to better understand the effects of PCBs on theeveloping cholinergic system (Eriksson et al., 2001). Interestingly,CNP precursor is ubiquitously expressed and both HCNP pre-

ursor and polypeptide can be measured in blood and plateletsGoumon et al., 2004). Blood samples may therefore provide aurrogate media to estimate contaminant-induced perturbation ofrain functions, assuming that brain and blood HCNP levels can beorrelated.

ters 184 (2009) 176–185 183

Exposure to MeHg has been shown to affect Tubulin polymeriza-tion and post-translational modification (Ishida et al., 1997; Miuraet al., 1984; Yagame et al., 1994). However, it is in PCBs and OCs treat-ment groups that an apparent shift between two Tubulin beta 2Bpost-translational states was detected. The last protein identifiedwas Ring finger protein 141. This transcription factor was origi-nally cloned from rat testis where it is thought to be involved inspermatogenesis (Qiu et al., 2003). However, in addition to a shorttranscript specifically expressed in testis, this gene also has a longertranscript expressed in the brain (Qiu et al., 2003). Transcriptionfactors are generally not among the most abundant cellular pro-teins and thus, identification of Ring finger protein 141 is rathersurprising and should be considered with caution until further con-firmation by other methods.

In conclusion, co-administration of up to 27 environmental con-taminants below their LOAEL (1× dose levels) did not result inmeasurable toxicity. At 100× dose levels, the effects of the North-ern Contaminant Mixture on pups growth and mortality wereattributed to MeHg, while effects on thyroid hormone systemwere mostly attributable to PCBs. However, PCBs and PTU-inducedhypothyroxinemia had very different effects on pup growth, serumTSH and brain taurine levels. Analysis of two-dimensional pro-tein electrophoresis patterns revealed that only a few proteinsdifferentially expressed in MeHg, PCBs and OCs treatment groupswere similarly affected following exposure to the NCM and hence,most of the effects of NCM on protein expression could not beaccurately predicted from the effects of the components adminis-tered separately. Proteins involved in mitochondrial and neuronalfunctions represented the majority of the differentially expressedproteins identified by MALDI-TOF/TOF MS. Immunological verifica-tion of protein identity confirmed that environmental contaminantmixtures affected protein post-translational modification states.Proteomic investigation also identified potential biomarkers andgenerated hypotheses whose validation will require further inves-tigations.

Conflict of interest

The authors have no conflict of interests to declare.

Acknowledgements

This study was funded in part by the Northern ContaminantsProgram from Indian and Northern Affairs Canada. The authorswant to thank Drs. Phil Shwed and Ivan Curran for critical reviewof this manuscript.

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