Naphthenic Acid Mixtures from Oil Sands Process-Affected Water

Jul 16, 2015 - Interestingly, exposure of undifferentiated mouse ESCs to the NA extract did not change the expression level of pluripotency markers (i...
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Naphthenic Acid Mixtures from Oil Sands Process-Affected Water Enhance Differentiation of Mouse Embryonic Stem Cells and Affect Development of the Heart Paria Mohseni,*,† Noah A. Hahn,†,∥ Richard A. Frank,‡ L. Mark Hewitt,‡ Mehrdad Hajibabaei,†,§ and Glen Van Der Kraak*,† †

Department of Integrative Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada Water Science and Technology Directorate, Environment Canada, Burlington, Ontario L7S 1A1, Canada § Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario N1G 2W1, Canada ‡

S Supporting Information *

ABSTRACT: Extraction of petrochemicals from the surface mining of oil sand deposits results in generation of large volumes of oil sands process-affected water (OSPW). Naphthenic acids (NA) are generally considered to be among the most toxic components of OSPW. Previous studies have shown that NAs are toxic to aquatic organisms, however knowledge of their effects on mammalian health and development is limited. In the present study, we evaluated the developmental effects of an NA extract prepared from fresh OSPW on differentiating mouse embryonic stem cells (ESC). We found that treatment of differentiating cells with the NA extract at noncytotoxic concentrations alters expression of various lineage specification markers and development of the heart. Notably, expression of cardiac specific markers such as Nkx2.5, Gata4, and Mef 2c were significantly up-regulated. Moreover, exposure to the NA extract enhanced differentiation of embryoid bodies and resulted in the early appearance of spontaneously beating clusters. Interestingly, exposure of undifferentiated mouse ESCs to the NA extract did not change the expression level of pluripotency markers (i.e., Oct4, Nanog, and Sox2). Altogether, these data identify some of the molecular pathways affected by components within this NA extract during differentiation of mammalian cells.

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OSPW.5 OSPW is associated with developmental defects in yellow perch embryos (Perca flavescens),6 Japanese Medaka embryos (Oryzias latipes)6 and two species of larval amphibian (Bufo boreas, Rana pipiens).7 Impaired growth has been described in mallard exposed to OSPW (Anas platyrhynchos).8 Moreover, OSPW has effects on the reproductive physiology of fathead minnow (Pimephales promelas)9,10 and goldfish (Carassius auratus)11 evident through reductions in plasma steroid concentrations and spawning success. Numerous studies have widely attributed the acid-extractable fraction of OSPW (which include NAs) to be its most toxic elements. Naphthenic acids are a water-soluble mixture of acyclic, monocyclic and polycyclic carboxylic acids that naturally occur in bituminous deposits.12 These petrogenic compounds are mobilized during caustic bitumen extraction and their high water solubility contributes to their elevated concentration in process-affected water where they can reach

INTRODUCTION An estimated 26.6 billion m3 of established crude bitumen reserves are present in Alberta, Canada and operations to extract bitumen in the Athabasca region are being expanded.1 This unconventional source of crude oil is accessible through either surface mining of superficial deposits (70% purity) at 300 and 900 mg/kg/d would cause a significant reduction in the number of live born pups. Moreover, alive pups born in the high dose group (i.e., 900 mg/kg/d) had a significantly lower body weight and decreased survival rate.18 However, the cause of embryonic and postnatal lethality remains to be investigated.18 Mouse embryonic stem cells, which are derived from the inner cell mass of blastocyst are pluripotent (i.e., capable of differentiating into all the three primary germ layers) and can grow indefinitely in vitro.19 These cells have been used to

model embryonic development in vitro and are amenable to studying cardiotoxicity, hepatotoxicity and neurotoxicity.20,21 One widely used method for differentiation of mouse ESCs involves formation of embryoid bodies (EBs).22,23 In this assay, ESCs are coalesced into 3-dimentional aggregates (i.e., EBs), which then spontaneously differentiate to all the three primary germ layers (endoderm, mesoderm, and ectoderm). This procedure aims to simulate aspects of cellular differentiation in the early embryo.22−24 Plating of EBs promotes further differentiation. Moreover addition of various growth factors to the culture media enables direction of differentiation pathways. In the presence of fetal bovine serum (FBS), cardiomyocyte lineage is one of the main differentiation pathways of the EBs. Subsequently this will result in appearance of morphologically distinct cardiac beating clusters. In the present study, mouse ESCs were used to examine the effect(s) of NA exposure on development. Our data suggest that exposure of differentiating mouse embryonic stem cells to the NA extract affects the specification process of the cells toward the cardiac and neural lineages.

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MATERIALS AND METHODS Naphthenic Acid Extraction/Source. The NAE used in this study was prepared according to the method described by Frank et al. (2006).25 In brief, acid extractable organics (AEOs) were isolated from freshly generated OSPW collected from the discharge into an active tailings pond in the summer of 2009. The OSPW was acidified to pH 2, the aqueous layer was decanted, and the precipitate (slurry) was centrifuged. The pellet was then dissolved in a 0.1 M NaOH solution before being passed through a bed of the weak anion exchanger diethylaminoethyl-cellulose to remove humic-like substances. The filtered NAE was washed with dichloromethane to remove neutral organics such as PAHs. The extract was again acidified, and the precipitated acids were collected on a 0.2 μm Teflon filter. The acid precipitate was then redissolved in 0.05 M NaOH and the concentration of this NAE stock solution (2504 10166

DOI: 10.1021/acs.est.5b02267 Environ. Sci. Technol. 2015, 49, 10165−10172

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Figure 2. Schematic representation of the experimental setup. Hanging drops (EBs) were made at d0 and were grown for 3 days. On the third day, EBs were collected and transferred to a suspension culture and were grown for an additional 2 days. EBs were collected at d5 and stored for RNA isolation. For beating cluster assay, after 5 days of growth, individual EBs were transferred to gelatinized 24-well plates, and their beating activity was assessed morphologically at day 7.

Beginning on day 7, beating activity of cardiomyocytes was assessed daily by visual inspection under light microscopy (Figure 2). Beating activity was recorded as the proportion of clusters beating from the total pool of EBs. In the short treatment experiment (d3−7), exposure of cells to the NAE started at day3 of differentiation. Two independent experiments (d0−7) were carried out for 0.0025 mg/L of NAE and 3 experiments for 2.5, 0.25, and 0.025 mg/L of NAE as well as the negative control group (i.e., not exposed to the NAE). Total number of EBs for each groups were as follows: nnegative control = 44, nNAE 2.5 mg/L = 40, nNAE 0.25 mg/L = 40, nNAE 0.025 mg/L = 48 and nNAE 0.0025 mg/L = 28. For short treatment (d3−7) two independent experiments were performed for 2.5 mg/L NAE and the negative control. Total number of EBS were as follows: nnegative control = 44, nNAE 2.5 mg/L = 51. In all experiments, exposure started at day 0 with undifferentiated mESCs. At various times during the differentiation protocol, EBs were harvested for RNA extraction. EBs were dissociated, washed with differentiation medium then washed with PBS. Cell pellets were stored at −80 °C until later use. Two independent experiments were carried out for each dilution of NAE and the negative control (i.e., not exposed to the NAE). Number of samples for each group were as follows: Experiment #1: nNAE negative control = 3, nNAE 2.5 mg/L = 5, NAE 0.25 mg/L n = 5, nNAE 0.025 mg/L = 3, and nNAE 0.0025 mg/L = 3. Experiment #2: nNAE negative control = 6, nNAE 2.5 mg/L = 6, nNAE 0.25 mg/L = 5, nNAE 0.025 mg/L = 6, and nNAE 0.0025 mg/L = 6. mESC Monolayer Cultures. mESCs were initially cultured as described above. To eliminate fibroblasts, the mESCs were serial passaged to a gelatinized dish for three passages. For NAs exposure, mESCs were cultured for 24 or 48 h in media containing LIF, harvested by trypsinization and frozen at −80 °C for subsequent measurement of gene expression using QPCR. RNA Purification, cDNA Synthesis, and Q-PCR. Total RNA from frozen cells was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany). mRNA was converted to cDNA using the commercially available SuperScript III First Strand Synthesis System (Invitrogen, California, U.S.A.) with 800 ng RNA loaded per reaction. Reaction mixes for Q-PCR contained 7.5 μL PerfeCTa SYBR Green I mix (Quanta Biosciences,

ug/mL) was determined using negative ion electrospray ionization mass spectrometry26 with nominal concentrations used for all subsequent dilutions and cell incubations. As there is a lack of representative analytical standard methodologies for the analysis of total AEOs or NAs, the results presented in this manuscript should be considered internally consistent, but may not be directly comparable to other results. For further information on the methods and analytical procedures used in this study, please contact the corresponding author. Mouse Embryonic Stem Cell (mESC) and Differentiation Culture. Undifferentiated R1 mESCs (F1 hybrid of 129 × 1 and 129S1 strains obtained from Laboratory of Dr. Andras Nagy, University of Toronto, Ontario, Canada) were cultured under standard conditions.19 In this case, mESCs were cocultivated with mitotically inactivated CF1 mouse embryonic fibroblasts (MEF; GlobalStem, Rockville, MD, U.S.A.) in high glucose (4.5 g/L) Dulbecco’s Modified Eagle Medium (Life Technologies, Carlsbad, NY, U.S.A.) containing 15% fetal bovine serum (HyClone, Waltham, Massachusetts, U.S.A.), 0.1 mM nonessential amino acids (Life Technologies), 2 mM glutamax (Life Technologies), 1 mM sodium pyruvate (Life Technologies), 0.1 mM 2-mercaptoethanol (Life Technologies), 10 000 U/mL penicillin-streptomycin (Life Technologies) and 10 ng/mL leukemia inhibitory factor27 (LIF; Life Technologies). Cells were maintained at 37 °C with 5% CO2. The mESCs were initially maintained on a layer of mitotically inactivated MEF to support their growth. Differentiation culture was initiated using the hanging drop technique.28 First, an undifferentiated mESC monolayer was suspended in differentiation medium, as above but lacking LIF and supplemented with 0.0025, 0.025, 0.25, and 2.5 mg/L of NAE (Figure 1). On day 0, 40 drops of 30 μL (800 cells/drop) were plated onto the lid of a bacterial dish (Greiner Bio, Frickenhausen, Germany). The lid was inverted onto a dish containing 10 mL PBS (Life Technologies) to maintain humidity, and incubated at 37 °C and 5% CO for 3 days. Hanging drop lids were washed with differentiation medium to collect embryoid bodies and seeded EBs were plated in low adhesion 6-well plates (Corning-Costar, Tewksbury, MA). After 2 days of incubation, individual EBs were collected and plated into separate wells of a gelatinized 24-well plate. 10167

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Figure 3. NAE affects expression of early cardiac markers in differentiating mouse ESCs. (a) Expression level of cardiac marker, Nkx2.5 is increased upon exposure of cells to 0.0025−2.5 mg/L NAE. (b,c) Gata4 and Mef 2c are up-regulated when cells are exposed to 0.025−2.5 mg/L NAE. No significant change was observed upon exposure to 0.0025 mg/L NAE. (d,e) Nrg1α and Nrg1β are up-regulated upon treatment of cells with 0.25−2.5 mg/L NAE. Treatment period = 5 days. Two independent experiments were carried out for each dilution of NAE and the negative control. Number of samples for each group were as follows: Experiment #1: nNAE negative control = 3, nNAE 2.5 mg/L = 5, nNAE 0.25 mg/L = 5, nNAE 0.025 mg/L = 3 and nNAE 0.0025 mg/L = 3. Experiment #2: nNAE negative control = 6, nNAE 2.5 mg/L = 6, nNAE 0.25 mg/L = 5, nNAE 0.025 mg/L = 6 and nNAE 0.0025 mg/L = 6. *p < 0.05.

50 μL of cells suspended in ESC media without LIF (500 cells) was seeded to a 96-well plate and incubated for 2 h to adhere to the surface. Then 150 μL of treated media was added to each well to generate a 10-fold dilution series of our stock NAE which ranged from 10 to 1 million fold dilutions. The highly cytotoxic compound 5-fluorouracil (0.06 mg/L) was used as a positive control. Media was changed on days 3, 5, and 7. On day 5 and 10 of culture, cells were incubated in PBS containing 5 mg/mL MTT. Mitochondrial dehydrogenases process MTT to a dark-blue, insoluble formazan product.28 MTT supernatant was aspirated and formazan was solubilized by the addition of isopropanol containing 0.7% [wt/vol] SDS. The plate was shaken for 15 min. Then OD570 of the supernatant was measured and corrected for background absorbance at 690 nm

Gaithersburg, MD, U.S.A.), 1.875 μL forward and reverse primers to a final concentration of 0.2 μM, and 3.75 μL of 10fold diluted cDNA. Q-PCR was performed using the PRISM 7000 platform (Applied Biosystems, California, U.S.A.) with reaction parameters repeated for 40 cycles as follows: 5 min denaturation at 95 °C, 30 s annealing and elongation at 60 °C, and melting of complementary strands and spectrophotometric measurement for 2 min at 50 °C. Cycle threshold values were normalized to GAPDH expression using the ΔΔCt method.29 Primer information is shown in Supporting Information (SI) Table S1. Cell Viability Assessment with MTT. Cell viability assessment by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was performed according to Seiler & Spielmann (2011).28 From a maintenance plate of R1 mESCs, 10168

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Figure 4. NAE treatment promotes differentiation of cardiomyocytes. (a) Exposure of differentiating mouse ESCs to 0.025−2.5 mg/L NAE for 7 days results in enhanced differentiation of cardiomyocytes. Exposure to 0.0025 mg/L NAE did not cause a significant difference. (b) Short treatment of EBS (i.e., d3 to d7) with 2.5 mg/L NAE resulted in increased number of beating clusters at day 7. At least two independent experiments were carried out for each dilution of the NAE and the negative control. Total number of EBs for each groups were as follows: long treatment (d0−7) nnegative control = 44, nNAE 2.5 mg/L = 40, nNAE 0.25 mg/L = 40, nNAE 0.025 mg/L = 48, and nNAE 0.0025 mg/L = 28. Short treatment (d3−7): n (d3−7) negative control = 44, n (d3−7)NAE 2.5 mg/L = 51.*p < 0.05.

increased number of beating clusters at day 7 of differentiation (Figure 4a). Moreover when the assay was performed with 0.0025 mg/L of the NAE there was no significant difference between the experimental and the control group (i.e., not exposed to the NAE) (Figure 4a). These observations corroborate our gene expression results. In order to provide evidence that the observed phenotype is not due to inferior performance of the selected cell line (i.e., R1) or batch of FBS, we continued the culture until day 10 and re-examined the nontreated EBs at that point. Majority of nontreated EBs (∼89%) were beating at day 10 suggesting that the low number of beating clusters at day 7 in the nontreated group is not due to the inability of cells to differentiate (SI Figure S2). Overall, these results demonstrate that exposure to the NAE affects early cardiac specification events, which control the heart development. NAE Does Not Impact Pluripotency Signaling Network in Embryonic Stem Cells. The accelerated differentiation could be the result of down-regulation of pluripotency network when cells are exposed to the NAE. In order to examine this possibility, we exposed undifferentiated mESCs to 2.5 mg/L of the NAE for 24 and 48 h and examined the expression levels of three pluripotency markers (Oct4 (Pou5f1), Nanog, and Sox2). In all experiments, exposure of mESCs to the NA extract did not affect the expression levels of Oct4, Nanog, or Sox2 (SI Figure S3a−c). These data suggest that the NAE does not affect the pluripotency network of mouse embryonic stem cells and the observed phenotype is not due to a general down-regulation of pluripotency genes. Further experiments were conducted to test whether an early stage of differentiation (i.e., exit from the pluripotent phase) was interrupted when the cells are exposed to the NA extract. In order to examine this scenario, we performed another beating cluster assay in which EBs were allowed to differentiate for 3 days with normal media. At day 3 of differentiation, EBs in the experimental group were exposed to 2.5 mg/L of the NAE, while the control group continued to differentiate in the normal media. The beating activities of the EBs in both groups were examined at day 7. Similar to our previous observations, exposure to 2.5 mg/L of the NAE enhanced the appearance of the beating clusters (Figure 4b). These data suggest that even a short treatment (i.e., 4 days) of cells with the NA extract is sufficient to interrupt the cardiac differentiation pathway.

(SpectaMax Plus 384, Molecular Devices, Sunnyvale, CA, U.S.A.). Statistical Analysis. Statistical analysis of gene expression data was performed using t test followed by the Bonferroni posthoc test. Values are presented as mean ± SE p < 0.05 was considered statistically significant. To compare the proportion of beating embryoid bodies Chi-square test was used.

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RESULTS Exposure to NAE Accelerates Differentiation of Embryonic Stem Cells into the Cardiac Lineage. One of the first events that occur during embryonic development is the formation of the cardiovascular system. In mammals, the heart is the first functional organ and as a result the cardiovascular system can be the target of environmental stressors that may happen in early stages of embryonic development. Our initial studies determined the effects of the NAE on cell viability. NAE at concentrations of up to 2.5 mg/L had no effect on cell viability as determined by the MTT assay (Figure 1 and SI Figure S1). To study the effects of the NAE on heart development, EBs were exposed from day 0 and RNA was extracted at day 5 (Figure 2). Subsequently, expression of master regulators of cardiac development (i.e., Nkx2.5 and Gata4) was determined by Q-PCR. Exposure of EBs to the NAE for 5 days caused a significant increase in the expression of Nkx2.5 at concentrations of 0.0025 to 2.5 mg/L (Figure 3a). In contrast, expression of a second cardiac marker Gata4 was unaffected by exposure to 0.0025 mg/L of the NAE but was increased in response to higher concentrations of NAE (0.025 to 2.5 mg/L; Figure 3b). In order to provide further evidence for enhancement of cardiomyocyte differentiation, we examined expression levels of additional early cardiac markers (i.e., Mef 2c, Nrg1α, and Nrg1β). Mef 2c was up-regulated in cells treated with 0.025 to 2.5 mg/L NAE while Nrg1α and Nrg1β were upregulated in response to 0.25 to 2.5 mg/L NAE (Figure 3c−e). To further investigate the effect of NA extract on differentiation and examine the proximate effects of the observed up-regulation of cardiac markers, a beating cluster assay was performed. Differentiating EBs were exposed to NA extract from day 0, and at day 7 of differentiation, beating profiles were examined morphologically (Figure 2). Exposure of differentiating EBs to 0.025 to 2.5 mg/L of the NAE resulted in 10169

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OSPW extracts demonstrated that fathead minnow embryos had declining embryonic heart rates with increasing NA exposure.16 Notably, our gene expression data indicate that development of the nervous system may also be affected by the exposure to the NAE, albeit at higher concentrations. More functional studies are required to examine the possible outcome and the phenotype, which may arise as the result of changes in expression level of early neural markers. More specific in vitro assays in which ESCs are directly differentiated toward the neural linage could shed light on the possible effects of the NAE on development of the nervous system. It is important to note that when ESCs are spontaneously differentiated via formation of embryoid bodies, cells of all the three primary germ layers (i.e., ectoderm, endoderm, and mesoderm) are formed. Therefore, each embryoid body is a heterogeneous population of various lineages. The complexity resulting from this lineage diversity hinders detailed analysis such as examining the potential effect on cardiac sublineages (e.g., myocardial, endocardial, and epicardial cell types) and the chemical dilution effect. This may also explain the differences we observed in up-regulation of Nrg1α and Nrg1β in response to various doses of the NAE. Our work was focused on the use of mouse ESC system for assessing the potential toxicity of this NAE in early mammalian development. Although our experiments were conducted to provide insights on cardiac and neural systems, our model can also be used through targeted tests and/or transcriptomic studies to provide a broader picture and insights as to what other organs may be affected by exposure to OSPW and NA extracts. Additionally, although mouse has been used as a model system to study mammalian development and mouse and human share many similarities in the pathways that control the major events happening during the embryonic development, it is important to note that there are differences between the two. Therefore, it is beneficial to assess the differentiation of human embryonic stem cells and examine the response of cardiac and neural pathways to the NAE in those cells. It would also be appropriate in future work to evaluate NA mixtures isolated from other “fresh” and “aged” industrial sources of OSPW, as well as bitumen-derived NA mixtures that occur naturally. In conclusion, our data indicate that exposure to the NAE utilized in this investigation affects the expression of key developmental markers in differentiating mouse ESCs. This may cause developmental abnormalities particularly in the heart and nervous system. These findings enhance our understanding of molecular mechanisms through which the components within complex NAE mixtures affect mammalian development and emphasize the importance of testing organisms during sensitive developmental stages in the environmental risk assessment of OSPW.

Together these results indicate that the observed phenotype is not the result of interruption in the pluripotency signaling pathways or defective exit from the pluripotent phase but a differentiation-dependent phenotype. NAE Treatment Leads to up-Regulation of Early Neuroectodermal Markers. Development of the nervous system is another major event that begins in early stages of mammalian development, hence making it a target for environmental stressors. In order to evaluate the possible effects of the NAE exposure on development of the nervous system we examined the expression levels of two early neuroectodermal markers (i.e., Nes and Sox1). As previously described, EBs were exposed to 0.0025−2.5 mg/L of the NAE from day 0 of differentiation and RNA was extracted at day 7. Relative expression levels of Nes and Sox1 were examined by QPCR, which revealed significant up regulation of both Nes and Sox1 at the highest concentration tested, 2.5 mg/L (SI Figure S4a,b). However, unlike the cardiac markers, when the concentration of the NAE was reduced to 0.0025−0.25 mg/ L, expression levels of Nes and Sox1 did not change significantly (SI Figures S4a,b). These results indicate that although the nervous system can be affected by the exposure to the NA extract, it may not be as sensitive as the cardiovascular system.

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DISCUSSION The results presented in this study show that exposure of differentiating mouse embryonic stem cells to an NAE from fresh OSPW causes significant changes in the expression of genes controlling the cardiovascular and nervous system development. Our data further suggest that the observed phenotype is not due to suppression of the pluripotency network but rather an event that occurs during the process of differentiation. ESCs have been used to study the effects of environmental toxicants on mammalian development.21,28,30,31 Recent advances in differentiation of ESCs to various lineages make them an attractive tool for exploring the molecular mechanisms of toxicant action and identifying the affected pathways. EBs closely mimic the events which occur in early stages of development in vivo. During embryogenesis, the cells follow a precise roadmap for differentiation. The balance between proliferating and differentiating cells and the time of differentiation are crucial for normal development of the organism. Our results show that exposure to the NAE affects differentiation of the developing cardiomyocytes as evident by upregulation of early cardiac markers and increased number of beating clusters at day 7. Premature differentiation hinders expansion of cardiac progenitors, which would otherwise continue to proliferate and contribute to the normal development of the cardiovascular system. This may ultimately result in a smaller number of cardiomyocytes. Our results are in-line with previous studies pointing to the developing heart as the target of environmental toxicants in fish.16,32−38 Exposure of zebrafish embryos to polycyclic aromatic hydrocarbons (PAHs) induces defects in formation of the cardiovascular system.32−34 Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in decreased number of cardiomyocytes, which ultimately leads to heart malfunction.35,36 Cardiac abnormalities also occur upon exposure of marine medaka (Oryzias melastigma) and zebrafish embryos to flame retardant additives, such as polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecanes (HBCDs).37,38 Moreover, recent work that utilized this same NAE as one of a series of

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ASSOCIATED CONTENT

* Supporting Information S

(Table S1) Primers used for Q-PCR; (Figure S1) cytotoxic effects of the NAE in mESC (short treatment); (Figure S2) quality control of mESC differentiation; (Figure S3) NAE does not affect the pluripotency network; and (Figure S4) early neuroectodermal markers are up-regulated upon exposure to NAE. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b02267. 10170

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(14) Kavanagh, R. J.; Frank, R. A.; Burnison, B. K.; Young, R. F.; Fedorak, P. M.; Solomon, K. R.; Van Der Kraak, G. Fathead minnow (Pimephales promelas) reproduction is impaired when exposed to a naphthenic acid extract. Aquat. Toxicol. 2012, 116−117, 34−42. (15) Rogers, V. V.; Wickstrom, M.; Liber, K.; MacKinnon, M. D. Acute and subchronic mammalian toxicity of naphthenic acids from oil sands tailings. Toxicol. Sci. 2002, 66 (2), 347−55. (16) Marentette, J. R.; Frank, R. A.; Bartlett, A.; Gillis, P.; Hewitt, L. M.; Peru, K.; Headley, J.; Brunswick, P.; Shang, D.; Parrott, J. Toxicity of naphthenic acid fraction components extracted from fresh and aged oil sands process-affected waters, and commercial naphthenic acid mixtures, to fathead minnow (Pimephales promelas) embryos. Aquat. Toxicol. 2015, 164, 108−17. (17) Garcia-Garcia, E.; Pun, J.; Perez-Estrada, L. A.; Din, M. G.; Smith, D. W.; Martin, J. W.; Belosevic, M. Commercial naphthenic acids and the organic fraction of oil sands process water downregulate pro-inflammatory gene expression and macrophage antimicrobial responses. Toxicol. Lett. 2011, 203 (1), 62−73. (18) McKee, R. H.; North, C. M.; Podhasky, P.; Charlap, J. H.; Kuhl, A. Toxicological assessment of refined naphthenic acids in a repeated dose/developmental toxicity screening test. Int. J. Toxicol. 2014, 33 (1), 168S−180S. (19) Behringer, R.; Gertsensten, M.; Vintersten, K.; Nagy, A. Manipulating the Mouse Embryo; A Laboratory manual. 4th ed. ed.; Cold Spring Harbor Press: Cold Spring Harbor, NY, 2013. (20) Kolaja, K. Stem cells and stem cell-derived tissues and their use in safety assessment. J. Biol. Chem. 2014, 289 (8), 4555−61. (21) van Dartel, D. A.; Pennings, J. L.; de la Fonteyne, L. J.; Brauers, K. J.; Claessen, S.; van Delft, J. H.; Kleinjans, J. C.; Piersma, A. H. Evaluation of developmental toxicant identification using gene expression profiling in embryonic stem cell differentiation cultures. Toxicol. Sci. 2011, 119 (1), 126−34. (22) Martin, G. R.; Wiley, L. M.; Damjanov, I. The development of cystic embryoid bodies in vitro from clonal teratocarcinoma stem cells. Dev. Biol. 1977, 61 (2), 230−44. (23) Doetschman, T. C.; Eistetter, H.; Katz, M.; Schmidt, W.; Kemler, R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 1985, 87, 27−45. (24) Sajini, A. A.; Greder, L. V.; Dutton, J. R.; Slack, J. M. Loss of Oct4 expression during the development of murine embryoid bodies. Dev. Biol. 2012, 371 (2), 170−9. (25) Frank, R. A.; Kavanagh, R.; Burnison, B. K.; Headley, J. V.; Peru, K. M.; Der Kraak, G. V.; Solomon, K. R. Diethylaminoethyl-cellulose clean-up of a large volume naphthenic acid extract. Chemosphere 2006, 64 (8), 1346−52. (26) Shang, D.; Kim, M.; Haberl, M.; Legzdins, A. Development of a rapid liquid chromatography tandem mass spectrometry method for screening of trace naphthenic acids in aqueous environments. J. Chromatogr A 2013, 1278, 98−107. (27) Chang, L.; Clifton, P.; Barter, P.; Mackinnon, M. High density lipoprotein subpopulations in chronic liver disease. Hepatology 1986, 6 (1), 46−49. (28) Seiler, A. E.; Spielmann, H. The validated embryonic stem cell test to predict embryotoxicity in vitro. Nat. Protoc. 2011, 6 (7), 961− 78. (29) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25 (4), 402−8. (30) Chen, X.; Xu, B.; Han, X.; Mao, Z.; Talbot, P.; Chen, M.; Du, G.; Chen, A.; Liu, J.; Wang, X.; Xia, Y. Effect of bisphenol A on pluripotency of mouse embryonic stem cells and differentiation capacity in mouse embryoid bodies. Toxicol. In Vitro 2013, 27 (8), 2249−55. (31) Wang, Y.; Fan, Y.; Puga, A. Dioxin exposure disrupts the differentiation of mouse embryonic stem cells into cardiomyocytes. Toxicol. Sci. 2010, 115 (1), 225−37. (32) Hicken, C. E.; Linbo, T. L.; Baldwin, D. H.; Willis, M. L.; Myers, M. S.; Holland, L.; Larsen, M.; Stekoll, M. S.; Rice, S. D.; Collier, T.

AUTHOR INFORMATION

Corresponding Authors

*Phone: (519) 824-4120, ext. 56213; e-mail: pmohseni@ uoguelph.ca (P.M.). *Phone: (519) 824-4120, ext. 53424; e-mail: gvanderk@ uoguelph.ca (G.V.D.K.). Present Address ∥

Department of Molecular Genetics, University of Toronto, Toronto, Ontario, M5S 1A8 Canada. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Irina Zdobnova and Jacquie Matsumoto for technical support.

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