Neurodevelopmental and Metabolomic Responses from Prenatal

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Neurodevelopmental and Metabolomic Responses from Prenatal Co-Exposure to Perfluorooctane Sulfonate (PFOS) and Methylmercury (MeHg) in Sprague-Dawley Rats Anthony J. F. Reardon, Jacqueline Karathra, Anton Ribbenstedt, Jonathan P. Benskin, Amy M. MacDonald, David W. Kinniburgh, Trevor J. Hamilton, Karim Fouad, and Jonathan W. Martin Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00192 • Publication Date (Web): 25 Jul 2019 Downloaded from pubs.acs.org on July 27, 2019

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Chemical Research in Toxicology

Neurodevelopmental and Metabolomic Responses from Prenatal Co-Exposure to Perfluorooctane Sulfonate (PFOS) and Methylmercury (MeHg) in Sprague-Dawley Rats

Anthony J.F. Reardon†, Jacqueline Karathra†, Anton Ribbenstedt‡, Jonathan P. Benskin‡, Amy M. MacDonald§, David W. Kinniburgh§, Trevor J. Hamilton║ Karim Fouad┴, Jonathan W. Martin†,,‡*

† Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Canada ‡ Department of Environmental Science and Analytical Chemistry (ACES), Stockholm University, Stockholm, Sweden § Alberta Centre for Toxicology, University of Calgary, Calgary, Canada ║ Department of Psychology, MacEwan University, Edmonton, Canada ┴ Department of Physical Therapy, University of Alberta, Edmonton, Canada

*Address correspondence to J.W. Martin, Department of Environmental Science and Analytical Chemistry, Stockholm University, Svante Arrhenius väg 8, SE-11418 Stockholm, Sweden Phone: +46 (0)72 146 2773, e-mail: [email protected]

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Abstract Methylmercury (MeHg) and perfluorooctane sulfonate (PFOS) are major contaminants of human blood that are both common in dietary fish, thereby raising questions about their combined impact on human development. Here, pregnant Sprague-Dawley rats ingested a daily dose, from gestational day 1 through to weaning, of either 1 mg/kg bw PFOS (PFOS-only), 1 mg/kg MeHg (MeHg-only), a mixture of 0.1 mg/kg PFOS and 1 mg/kg MeHg (Low-Mix), or of 1 mg/kg of PFOS and 1 mg/kg MeHg (High-Mix). Newborns were monitored for physical milestones and reflexive developmental responses, and in juveniles the spontaneous activity, anxiety, memory and cognition were assessed. Targeted metabolomics of 199 analytes was applied to sectioned brain regions of juvenile offspring. Newborns in the High-Mix group had decreased weight gain, as well as delayed reflexes and innate behavioural responses compared to controls and individual chemical groups indicating a toxicological interaction on early development. In juveniles, cumulative mixture effects increased in a dose-dependent manner in tests of anxiety-like behaviour. However, other developmental test results suggested antagonism, as PFOS-only and MeHgonly juveniles had increased hyperactivity and thigmotaxic behaviour, respectively, but fewer effects in Low-Mix and High-Mix groups. Consistent with these behavioural observations, a pattern of antagonism was also observed in neurochemicals measured in rat cortex, as PFOS-only and MeHg-only juveniles had altered concentrations of metabolites (e.g. lipids, amino acids, and biogenic amines), while no changes were evident in the combined exposures. The cortical metabolites altered in PFOS-only and MeHg-only exposed groups are involved in inhibitory and excitatory neurotransmission. These proof-of-principle findings at relatively high doses indicate the potential for toxicological interaction between PFOS and MeHg, with developmental-stage specific effects. Future mixture studies at lower doses are warranted, and prospective human birth cohorts should consider possible confounding effects from PFOS and mercury exposure on neurodevelopment.


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Introduction Perfluorooctane sulfonate (PFOS) and methylmercury (MeHg) are global environmental contaminants. PFOS is one of many perfluoroalkyl acids (PFAAs) that have been used directly as surfactants, or which are degradation products of precursor perfluoroalkyl substances used in surface treatment of textiles or food packaging. The widespread occurrence of PFOS in humans is attributable to its long historical use, environmental stability and persistence, and its lengthy blood elimination half-life (~5.4 yrs in humans)1. Although PFOS levels have declined in the blood of North Americans and Europeans since production was discontinued by the major manufacturer (3M company)2,3, it remains the predominant organic contaminant of human blood4,5 Although PFOS and its precursors were listed in the Stockholm Convention on Persistent Organic Pollutants in 20096, the exemptions under this treaty have allowed its continued production and use7 MeHg, a classic neurotoxicant8, is produced naturally through biological methylation of mercury and bioaccumulates in the aquatic foodweb8. Since the 1950s, a steady increase in Hg exported from the ocean has been observed that corresponds with increased catches from marine fisheries9. In humans, inorganic Hg has a relatively short half-life (~ 44 days)10,11 compared to PFOS. Chronic exposure to Hg is problematic for populations living near artisanal gold mining sites, or in coastal regions (e.g. Arctic or Mediterranean) where the diet includes major intake of seafood12. In general, dietary intake represents a major route of human exposure to both PFOS13–15 and MeHg16, and populations whose diet consists primarily of fish and seafood have higher exposure17,18. During pregnancy, both PFOS19 and MeHg20,21 efficiently cross the placenta into fetal circulation, with transfer efficiencies (ratio of maternal to fetal blood) ranging from 0.36 – 0.48 for PFOS22–24, and even higher (mean = 1.89) for MeHg20. Animal models have previously demonstrated both behavioural and molecular effects in offspring as a result of maternal MeHg exposure (see reviews by Johansson et al.25, Castoldi et al.26 and Bisen-Hersh et al.27). Gestational exposure results in offspring learning and memory deficits28–31 and decreased motor function32,33, and these MeHg-induced changes 3 ACS Paragon Plus Environment


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have been linked to altered brain morphology and neuron density28,34–37, as well as modified gene38 and protein39,40 expression. The neurodevelopmental effects of PFOS have received less attention, but chronic low-dose exposure to PFOS in pregnant rats induced altered open-field behaviour41,42 and motor function43,44 in offspring compared to controls. Evidence is also mounting in human populations, with studies demonstrating associations between PFAA exposure and fetal growth45–47 or motor and neurodevelopmental milestones in infants48. Cross sectional studies have revealed associations of various PFAAs including perfluorohexane sulfonate (PFHxS) and perfluorooctanoate (PFOA) along with PFOS, with increased incidence of attention deficit/hyperactivity disorder (ADHD) and impulsivity in school-age children49,50. Although MeHg and PFOS are major contaminants in background human populations, can act individually as developmental neurotoxicants, and both share a major pathway of exposure through diet, we are not aware that their combined effects have ever been investigated to date. Effects of chemical mixtures are difficult to interpret in epidemiological studies51, thus experimental animal models can be valuable for testing mixture interactions, whether independent, additive, synergistic or antagonistic. MeHg has been investigated in conjunction with other persistent organic pollutants with common exposure sources, such as polychlorinated biphenyls and organochlorines52–57. One study also investigated effects in offspring from co-exposure to MeHg and another PFAA, PFOA40. Metabolomics involves a comprehensive analysis of endogenous metabolites within a biological system to reveal alterations of biochemical pathways58, and has been utilized in developmental toxicity models of both fish59,60 and mice61. Perturbations in biochemical pathways help to reveal underlying mechanisms of altered behaviour as a result of developmental exposure to environmental contaminants. Here, the effects of dietary co-exposure to MeHg and PFOS were investigated in pregnant Sprague-Dawley rats and their offspring. Objectives were to determine if mixtures of PFOS and MeHg elicit different effects than corresponding individual exposures, and to determine if the mixture effect was different in a low- or high- dose of PFOS with the same dose of MeHg. Growth and development were monitored in offspring and behavioural (activity, anxiety, memory and cognition) outcomes were 4 ACS Paragon Plus Environment

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examined at pre-weaning and juvenile stages. Targeted metabolomics in distinct juvenile brain regions was used to investigate the underlying molecular mechanisms

Materials and Methods Animal treatment and dosing All protocols and procedures were approved by the Animal Care and Use Committee (animal use protocol, AUP #809) at the University of Alberta in compliance with guidelines of the Canadian Council on Animal Care and the Animal Protection Act (Government of Alberta, revised 2000). Sprague-Dawley rats (25 females and 12 males) were obtained from Charles River Laboratories (Laval, Quebec) and acclimated for 3 weeks prior to mating. The Sprague-Dawley strain, a multi-purpose model was selected based on its past use in both PFOS43,62,63 and MeHg64 developmental toxicology studies, as well as the availability of pharmacokinetic data. Animals were subjected to a 12 hr light/dark cycle and had unlimited access to food and water. Animals were bred by placing two females and one male overnight in a cage, after which pregnancy was confirmed by the appearance of a vaginal plug with the presence of sperm, denoted as gestational day (GD) 0. At GD 1, pregnant dams were randomly assigned to one of five treatment groups, and orally administered gelatin containing either MeHg, PFOS, combinations of both chemicals, or untreated gelatin (control). At birth, pups were culled to 8 animals/litter (5 female and 3 male), denoted as postnatal day (PND) 0. Dosing of maternal dams continued until weaning at PND 21. During pregnancy, dams were dosed according to treatment group. A stock solution of technicalgrade PFOS (Wellington Laboratories, Guelph ON) was prepared by dissolving the chemical in a 75:25 mixture of water and reagent alcohol (Sigma-Aldrich). Methylmercury chloride (CH3HgCl, SigmaAldrich) was dissolved in dimethyl sulfoxide (≥ 99.9% DMSO, Sigma-Aldrich). Stock solutions were further diluted to final concentrations of 0.4 % in commercial gelatin (strawberry-flavoured Jello®). Caution: CH3HgCl is hazardous and must be handled and stored according to guidelines provided in material safety data sheets from the manufacturer65. Doses were administered in a plastic cup to individual 5 ACS Paragon Plus Environment


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pregnant rats, with the amount of gelatin adjusted based on body weight (see SI for detailed information). The treatments were: Control (gelatin containing vectors from treatment groups), PFOS-only (1 mg PFOS/kg/d), MeHg-only (1 mg MeHg/kg/d), Low-Mix: (1 mg MeHg/kg/d + 0.1 mg PFOS/kg/d), and High-Mix (1 mg MeHg/kg/d + 1 mg PFOS/kg/d). The dose levels of MeHg and PFOS were selected based on previously published reports of the lowest observed adverse effect level (LOAEL) for offspring in chronic exposure studies of MeHg40,64,66,67 and PFOS62,68,69, to avoid maternal toxicity and neonatal mortality. Analysis of mercury and PFOS in dams and offspring PFOS was analyzed in the serum fraction of maternal and offspring rat whole blood samples. Samples were highly concentrated and diluted in LC-MS grade water (10, 100, 1000 and 10,000×) to the appropriate range for instrument detection. PFOS in diluted serum was extracted as described in Reardon et al. 201970, using a protein precipitation method modified from Glynn et al. 201271. Briefly, aliquots (0.5 mL) of each dilution containing an isotopically labeled PFOS standard (MPFAC-MXA; Wellington Laboratories) were extracted using 4 mL of acetonitrile (ACN). Samples were sonicated for 10 min in a room temperature water bath, followed by 5 min centrifugation at 2000 rpm (Eppendorf Sorvall ST-40R tabletop centrifuge, Thermo-Fisher Scientific). The supernatant was transferred to a 15 mL tube and evaporated in a 40 °C water bath under nitrogen gas to a volume of 0.2 mL, after which the extract was reconstituted to 1 mL in a 50:50 mixture of methanol and water. The diluted extract underwent dispersive cleanup as described by Powley et al. 200572, transferring the extract to a 1.7 mL Eppendorf tube containing approximately 0.025g of bulk graphitized carbon (Supelclean ENVI-Carb, Sigma Aldrich), that had been acidified with 50 µL of glacial acetic acid and mixed by vortex for 10 s. The sample was centrifuged for 10 min at 10,000 rpm (Sorvall Legend Micro 21R, Thermo Scientific) and the top 0.5 mL was transferred to an auto-sample vial. PFOS analysis with HPLC-MS/MS was performed according to the method described in Benskin et al. 201273, using a UFLC-XR Shimadzu HPLC coupled to an API 5000 triple quadrupole mass


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spectrometer (Applied Biosystems Sciex, Concord, ON). Total PFOS was calculated as the sum of its individual isomers (Linear-, 6m-, 5m-, ∑3m+4m-, 1m-, and ∑dimethyl-PFOS), quantified from an external solvent-based calibration curve spiked with native standard (BrPFOSK, Wellington Laboratories). Method validation procedures and results of recovery experimentation are presented in the supplementary information (Table S1). Rat whole blood was sent to the Alberta Centre for Toxicology for mercury analysis. A 100 µL aliquot of blood was diluted (20, 100, and 5000x) in volumes of LC/MS grade water. Samples were diluted further with a basic solution containing 25 µg/L of iridium internal standard, 10 µg/L gold, 0.5 g of EDTA in 1% v/v ammonia hydroxide, 2.5% butanol, and 0.05% v/v Triton X100. Treatment groups containing MeHg had basic solutions that were 2-fold more concentrated. The final diluents were then analyzed for total mercury by inductively coupled plasma mass spectrometry (ICP-MS/MS). QA/QC data from mercury analysis is available in the supplementary information (Tables S2-S4). Reproductive outcomes and growth rate of dams Various reproductive outcomes, including pregnancies carried to term, length of pregnancy, and litter size were monitored during the dosing period to assess reproductive success (Table S5). The weight of dams was recorded every 3 days during gestation (GD 1 to GD 19) and lactation (PND 2 to PND 21). Pre-wean observations and testing of newborn rat pups Every two days from PND 3 until PND 21, three pups were randomly selected from each litter of each group for observation and testing (Table 1). A modified test battery of reflex ontogeny was used for developmental testing of newborn rat pups74. Each pup was weighed, and monitored for developmental markers, including incisor eruption, hair growth, pinnae detachment, ear opening, and eye opening, as well as being subjected to a modified version of a functional test battery of reflex ontogeny74. Test responses were scored as, i) Righting reflex: the recorded time for the rat to place all four paws on a surface when initially placed on its back (cutoff time of 2 seconds), ii) Cliff drop aversion: the recorded time for the rat to retract its head and forepaws when placed on the edge of a tabletop with forepaws and 7 ACS Paragon Plus Environment


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head over the edge (cutoff time of 5 seconds) and iii) Negative geotaxis: the recorded time to turn 180⁰ when originally placed with head pointing downward on a 45⁰ slope (cutoff time of 5 seconds). Offspring post-wean behaviour Post-wean tests (Table 1) were initiated at PND 35 using 2 randomly selected females from each exposure group. Prior to testing, group labels were concealed and randomized, and tests were conducted by researchers that were blind to the exposure groups. An enclosed test space was allotted for mazes within the testing room to isolate animals from researchers, and all testing was conducted under uniform conditions of light (30 lux) and temperature (22 ⁰C). All mazes were built of black plexiglass (2 cm thickness) and animal activity was tracked by contrast (white rat on black background), recorded on an overhead camera and analyzed using Ethovision XT motion tracking software (v10, Noldus Information Technology, VA, USA). Open field arena Offspring within the open field (100 × 80 × 30 cm height), explored freely for a trial time of 5 min. Two consecutive trials were conducted with each rat, using a 24 hr inter-trial interval. Open-field activity was monitored, including total distance traveled (cm), mobility (sec), frequency (#), duration (sec), and latency of entry (sec). The software designated border and corner zones that were superimposed over the entire arena. The frequency (#) and duration (sec) of rearing activity were also recorded. Rotating rod (Rotarod) Offspring were tested on a single-lane commercial rotating rod apparatus (Med Associates, St. Albins, VT). A modification was applied to smooth the grooves of the axle (treadmill lane) to reduce the ability of the rats to cling to the rod and increased the difficulty of the task75,76. Rats were first subjected to a habituation phase for 2 min at 4 rpm for 1 hour prior to testing. The testing phase included single trial exposure to an accelerating speed (from 4 to 40 rpm) over 5 min77.


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Elevated plus maze From initial trials in the open field, animals displayed indications of anxiety-like behaviour (data not shown); consequently, a more thorough investigation of anxiety-like behaviour was carried out using the elevated plus maze. A commercial plus maze (Med Associates, St. Albins, VT) elevated at a height of 75 cm, consisted of two perpendicular open platforms (50 × 10 cm) and two perpendicular closed arms (50 × 10 × 40 cm) branching from a centre junction (12 × 12 cm). Rats were placed at the junction between the open and closed arms all facing the same direction and allowed 5 min for maze exploration. Total distance (cm) and tracked velocity (cm/sec), as well as frequency (#) and duration (sec) of arm entries was recorded. An index of anxiolytic activity was calculated, defined as an increase in the proportion of time spent in open arms divided by total maze time. Novel object recognition The novel object recognition test was modified from Ennaceur and Delacour78. Animals were previously habituated to the testing environment, as testing took placed 24 hrs after open field testing. The objects to be discriminated were yellow rubber ducks and black and white penguins. Multiple sets of objects were utilized and cleaned between trials to eliminate bias from olfactory stimuli, and separate naïve animals not involved in behaviour testing were used to verify that rats did not display any object preference. Testing consisted of two trials separated by an inter-trial time of 1 hour. The first trial (familiarization), allowed the animals to freely explore two identical objects placed in opposite corners of the arena for 10 min. A second trial (choice), replaced one of the familiar objects with a novel object, allowed animal exploration for 2 min. Exploration activity was defined as the animals’ head direction toward the object with nose proximity < 2 cm. The time of head direction facing toward, but not within proximity of the object was also recorded. The frequency (#), duration (sec), and latency (sec) of familiar and novel objects were recorded in addition to total exploration time. An object discrimination index, defined as the frequency or time exploring the novel object over total object exploration was calculated. 9 ACS Paragon Plus Environment


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Radial arm maze The radial arm maze consisted of a centre junction (30 cm in diameter) with 8 arms of equivalent dimensions (40 × 10 × 30 cm) branching out from the centre point. Food restrictions were implemented to increase rat motivation for rewards (banana flavoured pellets) during testing. Rats were habituated to the radial arm maze 24 hrs prior to training, each animal was given 5 min to explore an unbaited maze. The training phase with all arms baited was conducted for consecutive days until rats were able to navigate the maze without revisiting any target arms. The testing phase only baited 4 target arms and left 4 non-target arms unbaited. Rats freely explored the maze until all target arms were visited (maze completion) or until a threshold of 5 min. The testing was repeated for 4 consecutive days, recording the following parameters: Reference errors (non-target arm visits), and working memory errors (target arm revisits) as well as total errors were recorded for all rats. Tissue preparation, metabolite extraction and analysis Whole brains were removed immediately after euthanization and snap-frozen by submerging in isopentane kept on dry-ice. The time between extraction and snap-freezing was kept constant for all rats to minimize post-mortem associated fluctuations in metabolite levels. Frozen brains were kept on dry-ice, until partially thawed on ice, and brainstem, cerebellum, hypothalamus, hippocampus and cerebral cortex sections were carefully dissected. Sectioned samples were placed in 15 mL tubes and stored at -80 ⁰C. Frozen sections of brain were supercooled with liquid nitrogen, shattered into smaller pieces using a mortar and pestle, and weighed. Extractions were carried out by addition of solvent (1:4 chloroform:methanol) using 5 µL per mg tissue. Smaller brain sections (e.g. hippocampal sections) were diluted at 10 µL solvent/mg tissue to ensure sufficient volume for the extraction process. The solvent/tissue mixtures underwent disruption with beads using a 1600 MiniG automated tissue homogenizer (SPEX Sample Prep®) for 2 min at 1500 rpm (×2 intervals) using zirconium beads for soft tissue (hippocampus, hypothalamus, cortex), and steel beads for dense tissue (brainstem, cerebellum). Homogenates were centrifuged at 3000 RCF for 5 min in a tabletop centrifuge, after which the 10 ACS Paragon Plus Environment

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supernatant was removed and underwent ultracentrifugation at 12,000 RCF. The top 0.5 mL of extract was transferred to a 2.4 mL Eppendorf tube, after which 10 µL was transferred to a micro vial and combined with 60 µL of lipid internal standard, 20 µL of internal standard for targeted analysis of amino acids and biogenic amines and 210 µL of methanol. The targeted metabolomics method measures up to 199 metabolites, including, lipids, amino acids and biogenic amines, by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) with quantification by authentic standards. Analysis (i.e. UHPLC-hydrophilic interaction chromatography-MS/MS and flow-injection MS/MS) and nomenclature of amino acids (e.g. alanine = ala) and lipids followed methodology from Ribbenstedt et al79. A complete list of analytes, classes, abbreviations and internal standards and are found in the supplementary information (Table S6). Analysis of samples from dissected brain regions of rat offspring were randomized and run with intermittent procedural blanks and sequence quality control (QC) samples to minimize instrument carryover and to track instrumental drift over the course of the sequence. Blank signal subtraction was carried out for compounds detected in the procedural blank. Sequence drift correction was carried out using batchCorr80. Dissected brain regions of hypothalamus, hippocampus, cerebellum, brainstem, and cortex produced distinct metabolomic profiles (see Fig. S1 for a representative PCA plot of individual brain sections). Data handling and statistical analysis Statistical analysis of newborn and juvenile development outcomes utilized IBM SPSS statistics (version 24.0) with significance level of p < 0.05 in all cases. Pre-wean and post-wean observations and testing data were analyzed using non-parametric Kruskal-Wallis tests, followed by group comparisons using the Mann-Whitney test with an applied Bonferroni post-hoc correction (adjusted p-value). Consideration and examination of effects within and between litters is included in the supporting information.



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Statistical analyses of metabolomics data was carried out with Metaboanalyst 3.0 (www.metaboanalyst.ca)81. Analytes above the upper limit of quantification or below the limit of detection in brain section subgroups (e.g. Cortex) were not included in multivariate statistical analysis. Kruskal-Wallis testing identified significant (p < 0.05) differences in relative levels of metabolites between exposure groups within each dissected brain subsection. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were applied to data normalized with auto scaling (mean centered for each variable). PCA and PLS-DA scores plots indicated one outlier from each of the Control and Low-Mix groups, separate from the rest of the cluster within each treatment groups, this was confirmed with the outlier detection function of Random Forest testing, and outliers were subsequently removed from multivariate analysis. Hierarchal clustering of heatmap data utilized a Ward clustering algorithm and Euclidean distance measure. The pathway analysis feature of Metaboanalyst using the KEGG database identified specific pathways affected by PFOS and/or MeHg exposure. Pathway analysis uses a reference metabolome and provides the “pathway impact” if altered metabolites (listed in Table S6) are in key positions of the pathway network, and a “logP” indicating the level of significance of exposure induced deviations of metabolite concentrations from controls. In offspring, brain region-specific metabolite profiles were compared among controls, PFOSonly, MeHg-only, Low-Mix and High-Mix. Of 199 metabolites, 147, 136, 127, 132, and 138 were detected above detection limit in brainstem, cerebellum, cortex, hippocampus and hypothalamus, respectively. Only the cortical metabolomic profiles were considered as the cortex was the only tissue subsection that showed significant differences between treatment groups. PLS-DA model of cortical metabolites cross-validation produced a predictive (Q2) score of 0.8 and confirmed with permutation testing (p < 0.01), an indication of reproducibility and robustness of the model (Fig. S2).


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Results and Discussion Maternal and offspring exposure to PFOS and MeHg Analysis of plasma PFOS and blood total mercury of dams and offspring was measured at weaning (3 weeks post-birth). As expected, PFOS plasma concentrations of dams and offspring in the High-Mix group were similar to the PFOS-only group, and higher than the Low-Mix group. Also as expected, blood mercury content was consistent across all MeHg exposure groups (Figs. 1A and B, respectively). For both PFOS and mercury, significantly lower concentrations were observed in offspring compared to dams (5-fold and 40-fold lower, respectively) (Figs. 1A and B). The maternal-offspring transfer efficiency of PFOS was, therefore, greater than for MeHg (i.e. compare maternal High-Mix to offspring High-Mix, Fig. 1), yet both can be transferred transplacentally or through lactation. Animal models show a trend of decreasing blood and brain mercury levels in offspring during lactation66 and in infants during the first few months of breastfeeding in humans82. Breastmilk is a significant source of PFOS excretion for lactating mothers83 but contains low total mercury, primarily in the inorganic form84,85. The combination of decreasing transfer of MeHg throughout lactation and increasing body size of rapidly growing newborn pups may explain decreased mercury blood levels in rat offspring here. Maternal growth and reproduction There were no differences in gestation length or number of pups between exposure groups (Table S5), and no overt complications in pregnancy were noted. Survival rates of pups were 100 % in all litters. Maternal growth during gestation (GD0 to GD19), and lactation (PD 1 to PD 21) also did not show any treatment effect (Fig. 2A). Newborn offspring growth and reflex development Offspring weight gain was significantly decreased in both mixture exposure groups (Low-Mix and High-Mix, p < 0.05), whereas PFOS-only and MeHg-only offspring were not different from controls



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(Fig. 2B). Nevertheless, there were no significant differences in offspring achievement for many observed developmental milestones (Table 1). There were also no significant treatment differences in the proportion of newborns able to complete reflex ontogeny tasks (righting reflex, cliff aversion, or negative geotaxis, Fig. 3A-C, left column), however, a floor effect was observed for both cliff aversion (Fig. 3B) and negative geotaxis (Fig. 3C) up to PND 9, while a ceiling effect for both tasks was evident in the last week (i.e. > PND 15). Therefore, we restricted comparisons to specific days within the neurodevelopment window (Fig 3A-C, right column), and righting reflex and negative geotaxis were significantly delayed among High-Mix pups compared to controls (p < 0.05). In the cliff aversion task, High-Mix pups were also significantly delayed compared to control, PFOS-only, and MeHg-only on Day 11 (p < 0.05) (Fig. 3B). The pre-wean stage of newborn rats represents a sensitive stage of early neurological development during the brain growth spurt, occurring in the earliest postnatal period for rodents (PND 07) but in utero for humans (late 3rd trimester)86. The postnatal onset in rodents enables for observation of behavioural effects during stages of synaptogenesis, gliogenesis and myelination that is not possible in humans87. Decreased growth rates and induced delayed reflex responses in newborn rats in High-Mix pups did not occur in either PFOS-only or MeHg-only treatment groups (Figs. 2B and 3), indicating a mixture effect. Decreased growth and significantly delayed development of offspring has been observed in previous developmental toxicity studies of PFOS alone, but only at doses that caused increased neonatal mortally62. Previous animal co-exposure studies of MeHg and PFOA40, or of MeHg and polychlorinated biphenyls54 found that both the individual and combined exposures induced development delays, making it difficult to determine if a toxicological interaction was taking place. However, the current data show an interaction on newborn development, whereby significant effects were observed, but only from combined exposure to both PFOS and MeHg (High-Mix).


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Post-wean behaviour Behavioural outcomes of offspring exposed prenatally to PFOS, MeHg, or combined exposure are summarized in table 2. PFOS-only offspring were more active in the open field arena, with significantly increased travel distance, and velocity (Fig. 4A and B, respectively) compared to controls or the High-Mix group (p < 0.05). A visual representation of activity as heat maps (Fig. 4D) confirmed increased activity in the PFOS-only treatment. Offspring from both PFOS-only and MeHg-only treatments also had increased frequency of border crossings (Fig. 4C). Although PFOS-only and MeHgonly induced changes in offspring behaviour, these effects were absent in both combined exposure groups, suggesting an antagonistic toxicological interaction. PFOS-only offspring also performed better on the accelerating rotarod with significantly increase time to fall (p < 0.05) compared to control or HighMix groups (Fig. 5). PFOS-only offspring displayed hyperactivity in the open field (Fig. 4) that has been observed in previous studies of PFOS developmental toxicity in rats69, mice41,42, and zebrafish88,89. In zebrafish larvae, the PFOS-induced increase in activity was negated by administration of dopamine receptor agonists or amphetamine-like psychostimulants (e.g. dexafetamine, prescribed for treatment of ADHD), suggesting that the hyperactivity originates from altered levels of neurotransmitters. PFOS-only offspring also had increased performance on the rotarod (Fig. 5), a routinely administered test of motor coordination in rats and mice that was suggestive of improved motor function from PFOS exposure. However, rotarod performance may be influenced by the physical (i.e. level of activity) or mental (i.e. level of anxiety) state of the rat. For example, low doses of ethanol administered to mice increased their performance on the rotarod, whereas higher ethanol doses decrease performance90. Similar to low-dose ethanol, PFOS may have a stimulatory effect, increasing rat activity that manifests as increased time spent on an accelerating rotarod. When considered in conjunction with increased travel distance and velocity in the open field, the increased time to fall among PFOS-only rats was possibly attributable PFOS-related hyperactivity rather than improvement in motor function.



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In the elevated plus maze, High-Mix offspring had an increased number of entries (frequency), and a greater duration of the test time spent in open platforms compared to controls (Fig. 6A), resulting in an increased anxiety-index (p < 0.05), calculated as the proportion of time spent in open arms over the maze. An increased index represents an increased anxiolytic response, in other words decreased fear, and an observed increase in exploration. Further analysis of combined exposure groups revealed PFOS dosedependent changes in frequency, duration, and anxiety-index, as High-Mix offspring were significantly different from both the control and Low-Mix offspring (Fig. 6B). Control animals spent less time on open platforms (blue outline) and more time in closed arms (red outline) compared to both mixture exposure groups (Fig. 6C). Thus, increasing exposure to PFOS in the presence of MeHg corresponded to increased anxiolytic response on the elevated plus maze. In the open-field, MeHg-only and PFOS-only rats had increased border crossings compared to controls, but unlike PFOS-only, the MeHg-only observation did not coincide with increased distance or velocity, and instead may be attributable to increased thigmotaxic behaviour (Fig. 4). Thigmotaxis is part of the rats natural defense, avoiding predation by staying near to vertical surfaces91, a response that has been linked to anxiety-like behaviour92. MeHg-only offspring had increased border crossings but stayed in close-proximity with the walls of the arena, suggesting a thigmotaxic response and increased anxietylike behaviour. However, MeHg-only did not alter offspring behaviour on the elevated plus-maze (Fig. 6). Furthermore, increasing PFOS in the presence of MeHg (i.e. Low-Mix versus High-Mix) reduced anxiety in a dose-dependent manner, as these rats more freely explored the open platforms with increasing dose (Fig. 6). These findings suggest that maternal exposure to PFOS or MeHg alone is not enough to alter anxiety-related behaviour, but the combination of both contaminants with increasing PFOS had an interacting and cumulative effect. To our knowledge this is the only study to demonstrate a dose-dependent change in anxietyrelated behaviour from combined exposure to an organic contaminant with a heavy metal. Considering the anxiolytic nature of the observed response on the elevated plus maze, the reduced exploration (in the open field) may be attributed to MeHg-related impaired motor function. Our results indicated that 16 ACS Paragon Plus Environment

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offspring motor function on the rotarod was affected by prenatal exposure to MeHg (Fig. 5) but was not significant after Bonferroni adjustment. However, this finding is nevertheless supported by past studies of MeHg-induced decreases in rat motor function52,93,94, specifically decreased time to fall on the rotarod33,39,40,66. Offspring underwent two types of memory testing, a 1-day object recognition test of non-spatial oriented memory, and a multi-day test of spatial memory using the radial arm maze. None of the treatment groups were different from controls on tasks of non-spatial (e.g. novel object recognition) or spatial (e.g. radial arm maze) learning or memory (data not shown), agreeing with most studies of repeated low-dose exposure to prenatal MeHg exposure in rodents29,39,40,54,95,96. Thus, MeHg was not sufficient to induce cognitive deficits, even when combined with PFOS in the current study. Although there is extensive human evidence of developmental neurotoxicity from prenatal exposure to MeHg97, in rodent models the patterns of cognitive deficits are less consistent (see Castoldi et al. 200826 and BisenHersh et al. 201427 for review). Altered metabolite profiles in offspring All dissected brain regions (cortex, cerebellum, hippocampus, hypothalamus and brainstem) had distinct metabolite profiles (Fig. S1). However, only for the cortex were distinct profiles observed between treatment groups, with 68 significant alterations among 127 detected metabolites (p < 0.05, Fig. 7). Moreover, the antagonism between PFOS and MeHg observed at the behavioural level was reinforced by the metabolite profiles. For example, metabolite profile clusters in PLS-DA scores plot of PFOS-only and MeHg-only were distinct from controls, but both combined exposure groups (Low-Mix and HighMix) overlapped with controls (Fig. 7A). Within the cortex, 53.5 % of variance between treatments were explained by the first two components of the model with the majority on the x-axis (component 1) that accounted for 47.6 % (Fig. 7A). The loadings plot identified the lipids (primarily phosphatidylcholines) and specific metabolites that made the greatest contribution to treatment separation in the PLS-DA scores plot (Fig. 7B).



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Unsupervised hierarchal cluster analysis (heatmap, Fig. 7C) portrayed the extent of change in cortex metabolite response (rows) between individual treatment groups (columns). A distinct cluster was observed for PFOS-only and MeHg-only, and another cluster for control and both combined exposure groups. For amino acids and GABA, significantly higher concentrations were observed in MeHg-only or PFOS-only compared to controls or combined exposure groups. The opposite trend was observed for lipids, with higher concentrations observed in control and combined exposure groups, relative to MeHgonly or PFOS-only groups (Fig. 7C). Perturbation of cortical lipids (primarily phosphatidylcholines and sphingomyelins) in the MeHgonly and PFOS-only treatments (Fig. 7) are consistent with previous findings for PFOA61. Phosphatidylcholines in lipid membranes are reservoirs for choline, a precursor to acetylcholine, one of the primary neurotransmitters of the cholinergic system98. In mice, hyperactivity from exposure to PFOA and PFOS was altered by injections of nicotine41. Numerous investigations have also shown the capacity of PFAAs to affect expression of the alpha isotype of peroxisome proliferator activated receptors (PPARs)99. PPARα is primarily expressed in the liver, heart and skeletal muscle, and plays a key role in lipid metabolism100. However, PPARα is also strongly expressed in the prefrontal cortex of rodents and humans101. It is reasonable then, to postulate that exposure to PFOS, and to a lesser extent MeHg, affects lipid metabolism through altered PPARα expression, leading to decreased concentrations of phosphatidylcholines and disruption of cholinergic neurotransmission that ultimately impact behavioural outcomes. Levels of specific amino acids and neurotransmitters (Fig. 7B, green dashed circle) influenced the separation of clusters in the scores plot. Concentrations of GABA, taurine, Gly, Met, Pro, Ser, and T4hydroxyproline were increased with PFOS-only, as were concentrations of Thr and Ser in MeHg-only cortex, compared to controls (p < 0.05, Fig. S3). Metabolic pathways involving Gly, Ser, Thr, Ala, Asp, Arg, Pro, and Glu, were significantly affected by individual exposure to either PFOS or MeHg, while no significant changes were observed in either mixture group. The alterations of amino acids and neurotransmitters and their subsequent effects on metabolic pathways indicate target neurotransmission 18 ACS Paragon Plus Environment

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systems that are susceptible to change from early-life exposure to PFOS or MeHg and these are likely linked to the corresponding changes in behaviour. Metabolites are involved in a variety and physiological processes and provide potential targets of neurotoxicity from PFOS or MeHg exposure. Neurotransmitters GABA and Gly (Fig. S3), and their corresponding metabolic pathways (Fig. 8), were significantly affected by exposure to either PFOS- or MeHg-only. MeHg alters the activity of GABAα receptors, blocking GABAergic synaptic responses, inducing a hyper-excitability of neurons and increasing susceptibility to seizures102. In addition to GABA, neonatal exposure to MeHg alters locomotor activity mediated by the dopaminergic system103; as activity of dopamine D2 receptors in the striatum of rats were reduced from MeHg exposure, ultimately decreasing locomotor activity, an effect remedied by D2 receptor agonists93,94. PFOS alters the concentration and gene expression of both D1 and D2 dopaminergic receptors in prefrontal cortex, hippocampus and amygdala brain104. In our study, dopamine levels were below instrument detection limits and we were unable to observe differences between treatment groups. However, Asp, Gly, and Ser, agonists of N-methyl-D-aspartate (NMDA) receptors, were significantly elevated from PFOS exposure (Fig. S3). NMDA, a glutamate receptor that interacts with dopamine receptors modulates a variety of cognitive and motor functions in the brain105. Increased excitatory neurotransmission from activation of NMDA receptors provide a basis for increased spontaneous activity in PFOS exposed rats, as well as provide an explanation for concomitant feedback increases of the inhibitory neurotransmitter GABA. Interpretation of outcomes in combined exposures to PFOS and MeHg In previous animal studies, both MeHg-only and the combined exposure group (i.e. with PFOA) both elicited similar effects, making it difficult to determine if an interaction was taking place40,54. Here, significant effects on growth and development were only observed in newborn rat pups exposed to a mixture of PFOS and MeHg (i.e. Low-Mix and/or High-Mix), strongly suggesting an additive toxicological interaction between these two common contaminants (Figs. 2B and 3). Further studies are



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warranted to determine the threshold for this effect, as the current doses are not environmentally relevant but were chosen to allow fewer animals to be used in the current work. In older juveniles, PFOS and MeHg alone altered activity of rats in the open field and on the rotating rod, but this was not detected in either mixture exposure groups, indicative of antagonism, effects that were mirrored when comparing metabolomic profiles in the rat cortex. Such antagonism in juveniles may not be an indication of recovery from the initial additive adverse effects on newborn weight gain and early developmental responses. Changes induced from combined exposure in newborns were not readily observed at the later stages of adolescence or adulthood, indicating that interacting effects from contaminant mixtures may manifest differently over subsequent developmental stages. These findings reinforce the need for repeated measures and developmental testing in animal models as well as epidemiological studies to fully elucidate the effects from contaminant mixtures. The current observations of antagonism are consistent with past studies of MeHg with either PFOA40 or polychlorinated biphenyls53–56, where it has been suggested that organic contaminants have the capacity to mask effects of MeHg. For example, Coccini et al. found that relatively high doses of PCB153 affected the influence of MeHg on cholinergic muscarinic receptors. Given that both compounds have different molecular targets it was suggested that combined exposure may result in conformational changes in MeHg binding sites, thereby preventing its effects56. The antagonism demonstrated from combined exposure in our results exemplifies the need for inclusion of both compounds in epidemiological studies of either PFOS or Hg, as the results may be confounded if either of these contaminants is not adjusted for, or included as an interaction term in multivariate models.

Conclusion Chronic low-level exposure to both PFOS and MeHg sources elicits significant effects in early development, whereas only effects from individual exposure were observed at later stages. In juveniles, outcomes from behaviour testing and metabolomic analysis indicated that the combined effect of PFOS and MeHg mixtures were antagonistic, a finding consistent with previous observations53,54,96, indicating 20 ACS Paragon Plus Environment

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that co-exposure may not exacerbate behavioural or neurochemical effects from developmental exposure to either PFOS or MeHg individually. However, whether juveniles recovered from initial effects as newborns remains unclear. It is reasonable to postulate that underlying neurotoxic effects elicited during fetal and newborn development may persist into adulthood and more detailed investigations of specific altered metabolites and neurotransmitters (e.g., GABA) and their corresponding pathways are warranted to examine the extent of combined effects from chemical mixtures such as MeHg and PFOS.



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Author Information Corresponding Author J.W. Martin, Department of Environmental Science and Analytical Chemistry, Stockholm University, Svante Arrhenius väg 8, SE-11418 Stockholm, Sweden. Phone: +46 (0)72 146 2773, e-mail: [email protected] Funding Funding from the Canadian Institutes of Health Research (PI Martin, Application Number 275989) is acknowledged for making this research possible. A.J.F.R additionally acknowledges generous stipend funding from the Stollery Children’s Hospital Foundation and supporters of the Lois Hole Hospital for Women through the Women and Children’s Health Research Institute. Notes The authors declare that they have no competing financial interests

Acknowledgements We would like to thank the staff of the Health Sciences Laboratory Animal Services at the University of Alberta for their contribution and support to this project.

Abbreviations MeHg, methylmercury; PFAA, perfluoroalkyl acid; PFOS, perfluorooctane sulfonate; PFHxS, perfluorohexane sulfonate; PFOA, perfluorooctanoate; ADHD, attention deficit-hyperactivity disorder; GD, gestation day; PND, postnatal day; LOAEL, lowest observed adverse effect level; ACN, acetonitrile; (U)HPLC-MS/MS, (ultra) high performance liquid chromatography-mass spectrometry; ICP-MS/MS, Inductively coupled plasma-mass spectrometry; PCA, principal component analysis; PLS-DA, partial least squares discriminant analysis; NMDA, N-methyl-D-aspartate.


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Supporting Information 

Animal dosing methodology; Quality assurance and quality control data including detection and reporting limits, and recovery and precision of PFOS and MeHg; Litter effects and repeated measures; reproductive outcomes of pregnant dams exposed to PFOS and MeHg; analysis and listing of targets within metabolomics platform (including KEGG ID), description of nomenclature and supplementary figures identifying significantly altered metabolites, and crossvalidation and permutation testing for PLS-DA model validation



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Table 1. Timing of pre- and post-wean observations and testing of rat pups. Parameter

Post-Natal Day (PND) Growth 3 – 19 a Incisor eruption 3 – 19 a Hair growth 3 – 19 a Eyelid opening 3 – 19 a Pinnae detachment 3 – 19 a Ear opening 3 – 19 Righting reflex 3 – 19 Cliff drop aversion 3 – 19 Negative geotaxis 3 – 19 Open Field 38 – 39 Rotating Rod 40 – 41 Novel object recognition 42 Elevated plus maze 43 Radial arm maze 53 – 61 a observation representing developmental milestone


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Table 2. Summary of behavioural outcomes affected in rat offspring prenatally exposed to PFOS-only, MeHg-only, or mixtures of PFOS and MeHg. Outcomes are represented as significantly (p < 0.05) increased (↑) or decreased/delayed (↓). Black arrows correspond to changes in comparison to controls, and red arrows to changes from both control and High-mix treatment groups. Green shaded regions are evidence for a significant mixture-effect (synergism or additivity), whereas red shaded regions are evidence for antagonism. Apparatus and Test Parameters

PFOS-only

MeHg-only

Low-Mix

High-Mix

Pre-wean testing (newborns) growth weight

-

-

↓

↓

righting reflex

-

-

-

↓

cliff aversion

-

-

-

↓

negative geotaxis

-

-

-

↓

distance travelled

↑

-

-

-

velocity

↑

-

-

-

border crossing

↑

↑

-

-

performance

↑

↓b

-

↑a

Post-wean testing (juveniles) Open-Field

Rotating Rod Elevated Plus Maze

anxiety Index

-

-

↑a,b

Novel Object Recognition Radial Arm Maze

non-spatial memory

-

-

-

-

-

-

-

-

spatial-oriented memory a response was observed to be dose-dependent b no longer significant after Bonferroni correction



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Figure legend Figure 1. Concentration (mean ± SE) in serum (PFOS) and whole blood (mercury) of control (n = 5) and various treated (each n = 4), collected at 3-weeks parturition from dams (A), and offspring (B). Figure 2. Growth rates (mean ± SE) of (A) dams (n = 4 to 5), and (B) offspring (n = 16 to 20). Dams were monitored continuously; a break indicates the separation of measurements pre- and post-natal. Figure 3. Pre-wean growth and performance of newborn offspring in tests of (A) righting reflex, (B) cliff drop aversion, and C) negative geotaxis (n = 12 to 15). The proportion of pups able to complete the task is shown in the left panels, while time taken to complete the task is shown in the right panels (mean ± SE). Figure 4. Activity of offspring over 5 min exploration in the open field arena among 5 treatment groups (n = 8 each), showing (A) distance travelled, (B) velocity, (C) border frequency, and (D) heatmaps of activity (merged group mean). Figure 5. The recorded time to fall (mean ± SE) of offspring on the accelerating rotating rod from 5 to 40 rpm over 5 min for control and treatment groups (n = 8). Figure 6. Offspring anxiety-related behavioural activity on the elevated plus maze in controls compared to various treatments (n = 8). In the top panel (A) we test the hypothesis that the combination of (MeHg+PFOS) gives a different result from (PFOS-only) or (MeHg-only). In the bottom panel (B) we test the hypothesis that the dose of PFOS (Low or High) results in a difference at a constant dose of MeHg. Data shown (mean ± SE), includes the cumulative frequency of arm entries (left panels), the cumulative arm duration (middle panels), and a calculated anxiety index ratio of the proportion of time spent in open vs. closed arms (right panels). Panel (C) shows heatmaps of mean group activity with wall enclosed arms (red outline), and open-armed platforms (blue outline). Figure 7. A) PLS-DA scores plot, B) loadings plot, and C) hierarchical cluster analysis of significant metabolites contributing to the PLS-DA model quantified in offspring cortex (n = 5 per treatment) with biogenic amines and amino acids with the greatest influence highlighted with a dashed green circle. Metabolites (68 total) from PFOS-only (purple) and MeHg-only (teal) groups were significantly different from Control (red) and the combined exposure groups Low-Mix (green), and High-Mix (blue). A colour gradient shows metabolite-specific responses, representing increasing (red), and decreasing (blue) or no change (white) in concentration between treatment groups. Figure 8. Metabolic pathways influenced by altered concentrations of biogenic amines and amino acids in (A) MeHg-only and (B) PFOS-only offspring cortex. Metabolic pathways are identified as slightly (yellow), moderately (orange) or significantly (red) altered based on level of significance (log(p), y-axis), as well as the number of metabolites involved (increasing size).


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Figure 1. Concentration (mean ± SE) in serum (PFOS) and whole blood (mercury) of control (n = 5) and various treated (each n = 4), collected at 3-weeks parturition from dams (A), and offspring (B). 177x76mm (600 x 600 DPI)

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Figure 2. Growth rates (mean ± SE) of (A) dams (n = 4 to 5), and (B) offspring (n = 16 to 20). Dams were monitored continuously; a break indicates the separation of measurements pre- and post-natal. 155x67mm (600 x 600 DPI)


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Figure 3. Pre-wean growth and performance of newborn offspring in tests of (A) righting reflex, (B) cliff drop aversion, and C) negative geotaxis (n = 12 to 15). The proportion of pups able to complete the task is shown in the left panels, while time taken to complete the task is shown in the right panels (mean ± SE). 152x108mm (600 x 600 DPI)



Figure 4. Activity of offspring over 5 min exploration in the open field arena among 5 treatment groups (n = 8 each), showing (A) distance travelled, (B) velocity, (C) border frequency, and (D) heatmaps of activity (merged group mean) 151x98mm (600 x 600 DPI)


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Figure 5. The recorded time to fall (mean ± SE) of offspring on the accelerating rotating rod from 5 to 40 rpm over 5 min for control and treatment groups (n = 8). 84x64mm (600 x 600 DPI)



Figure 6. Offspring anxiety-related behavioural activity on the elevated plus maze in controls compared to various treatments (n = 8). In the top panel (A) we test the hypothesis that the combination of (MeHg+PFOS) gives a different result from (PFOS-only) or (MeHg-only). In the bottom panel (B) we test the hypothesis that the dose of PFOS (Low or High) results in a difference at a constant dose of MeHg. Data shown (mean ± SE), includes the cumulative frequency of arm entries (left panels), the cumulative arm duration (middle panels), and a calculated anxiety index ratio of the proportion of time spent in open vs. closed arms (right panels). Panel (C) shows heatmaps of mean group activity with wall enclosed arms (red outline), and open-armed platforms (blue outline). 152x108mm (600 x 600 DPI)


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Figure 7. A) PLS-DA scores plot, B) loadings plot, and C) hierarchical cluster analysis of significant metabolites contributing to the PLS-DA model quantified in offspring cortex (n = 5 per treatment) with biogenic amines and amino acids with the greatest influence highlighted with a dashed green circle. Metabolites (68 total) from PFOS-only (purple) and MeHg-only (teal) groups were significantly different from Control (red) and the combined exposure groups Low-Mix (green), and High-Mix (blue). A colour gradient shows metabolite-specific responses, representing increasing (red), and decreasing (blue) or no change (white) in concentration between treatment groups. 177x173mm (300 x 300 DPI)



Figure 8. Metabolic pathways influenced by altered concentrations of biogenic amines and amino acids in (A) MeHg-only and (B) PFOS-only offspring cortex. Metabolic pathways are identified as slightly (yellow), moderately (orange) or significantly (red) altered based on level of significance (log(p), y-axis), as well as the number of metabolites involved (increasing size). 170x85mm (300 x 300 DPI)


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For TOC only 82x44mm (600 x 600 DPI)


Neurodevelopmental and Metabolomic Responses from Prenatal

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