Arsenic and Its Methylated Metabolites Inhibit the Differentiation of

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Arsenic and Its Methylated Metabolites Inhibit the Differentiation of Neural Plate Border Specifier Cells Christopher R. McCoy,† Bradley S. Stadelman,‡ Julia L. Brumaghim,‡ Jui-Tung Liu,§ and Lisa J. Bain*,†,§ †

Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, South Carolina 29634, United States Department of Chemistry, Clemson University, 219 Hunter Laboratories, Clemson, South Carolina 29634, United States § Environmental Toxicology Graduate Program, Clemson University, 132 Long Hall, Clemson, South Carolina 29634, United States ‡

S Supporting Information *

ABSTRACT: Exposure to arsenic in food and drinking water has been correlated with adverse developmental outcomes, such as reductions in birth weight and neurological deficits. Additionally, studies have shown that arsenic suppresses sensory neuron formation and skeletal muscle myogenesis, although the reason why arsenic targets both of these cell types in unclear. Thus, P19 mouse embryonic stem cells were used to investigate the mechanisms by which arsenic could inhibit cellular differentiation. P19 cells were exposed to 0, 0.1, or 0.5 μM sodium arsenite and induced to form embryoid bodies over a period of 5 days. The expression of transcription factors necessary to form neural plate border specifier (NPBS) cells, neural crest cells and their progenitors, and myocytes and their progenitors were examined. Early during differentiation, arsenic significantly reduced the transcript and protein expression of Msx1 and Pax3, both needed for NPBS cell formation. Arsenic also significantly reduced the protein expression of Sox 10, needed for neural crest progenitor cell production, by 31−50%, and downregulated the protein and mRNA levels of NeuroD1, needed for neural crest cell differentiation, in a time- and dose-dependent manner. While the overall protein expression of transcription factors in the skeletal muscle lineage was not changed, arsenic did alter their nuclear localization. MyoD nuclear translocation was significantly reduced on days 2−5 between 15 and 70%. At a 10-fold lower concentration, monomethylarsonous acid (MMA III) appeared to be just as potent as inorganic arsenic at reducing the mRNA levels Pax3 (79% vs84%), Sox10 (49% vs 65%), and Msx1 (56% vs 56%). Dimethylarsinous acid (DMA III) also reduced protein and transcript expression, but the changes were less dramatic than those with MMA or arsenite. All three arsenic species reduced the nuclear localization of MyoD and NeuroD1 in a similar manner. The early changes in the differentiation of neural plate border specifier cells may provide a mechanism for arsenic to suppress both neurogenesis and myogenesis.

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INTRODUCTION Arsenic is a naturally occurring element found in bedrock, which has resulted in the contamination of groundwater in many parts of the world.1,2 In the West Bengal region of India, it is estimated that more than 26 million individuals have been exposed to drinking water contaminated with inorganic arsenic (As).3 Although the World Health Organization recommends a maximum concentration of arsenic in drinking water of 10 μg/L, some wells in West Bengal have levels up to 531 μg/L As.4 Inorganic arsenic has also been found as a contaminant in food, such as rice, with levels ranging from 0.15 to 0.36 mg/kg.5,6 In particular, increased inorganic and methylated arsenic levels in the urine of pregnant women and children have been seen due to the consumption of rice products.7 Once inside the body, inorganic arsenic can be methylated by arsenic methyltransferases, with monomethylarsonous acid (MMA III) and dimethylarsinous acid (DMA III) being the primary species excreted in the urine.8 It is known that the concentrations of MMA III and DMA III increase during pregnancy.9−11 In vitro studies have shown that some arsenic metabolites, © XXXX American Chemical Society

especially the trivalent species, are more toxic than inorganic arsenic itself. For example, MMAIII inhibited the differentiation of mouse ES-D3 stem cells into cardiomyocytes at much lower concentrations than arsenic trioxide or DMA III.12 Since there is an increase in arsenic biotransformation and methylated metabolites during pregnancy, continuous exposure to these metabolites could also have adverse effects on the developing fetus. Many adverse developmental outcomes can arise from being exposed to arsenic. Epidemiological studies have shown that in utero exposure to arsenic can lead to increased occurrence of neonatal death, low birth weight, and miscarriages.9,10,13,14 These outcomes are thought to result from arsenic’s ability to cross the placental barrier and come in contact with the embryo.9,15 For example, cord blood and placenta contained an average of 9 and 34 μg/L total arsenic, respectively, in women drinking water containing 200 μg/L of arsenic.9 Embryonic Received: January 21, 2015

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DOI: 10.1021/acs.chemrestox.5b00036 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology

Figure 1. Arsenic decreases Pax3 intensity and Msx1 and MyoD nuclear localization in day 2 embryoid bodies. After exposure to 0, 0.1, and 0.5 μM arsenic for 2 days, embryoid bodies were fixed, embedded, and stained for transcription factor expression. Representative images are shown (A, n = 3 per group). Intensity values were averaged and are presented as relative fluorescence after normalization to the control group ± standard deviation (B). For each image, cells (n = 100) were examined for nuclear localization of each marker, and the results are presented as % nuclear localization ± standard deviation (C). Statistical differences were determined by ANOVA followed by Tukey’s test (*, p < 0.05). Although NeuroD1 and myogenin expression were examined, they are not expressed until days 4 and 5, respectively.

NPB region, which induces the presumptive NC cells to become competent to the neural crest specifier signals.25 These cells will then undergo an epithelial to mesenchyme transition (EMT), delaminate, and migrate away from the neural tube.26 The signals that induce the NC come from genes that are collectively known as the neural plate border specifiers (NPBS).25 The signals include many transcription factors required for neurogenesis, such as Pax3, Sox10, and NeuroD,25 and required for myogenesis, such as Msx1, MyoD, and myogenin.27 The objective of this study was to determine whether arsenic specifically impairs the differentiation of neural plate border specifier cells into sensory neurons and skeletal muscle cells. Further, we wanted to examine whether the methylated metabolites of arsenite differed from inorganic arsenite in their ability to reduce cell differentiation. Our results suggest that early exposure to arsenite and its methylated metabolites impairs the differentiation of neural plate border specifier cells by reducing the expression of Pax3, Sox10, and NeuroD1 in the neurogenic pathway and altering the nuclear translocation of MyoD in the myogenic pathway.

arsenic exposure has also been correlated with many neurological deficits, such as decreased pattern memory, lower IQ scores, and mental retardation.16−20 Collectively, these studies suggest that arsenic impacts the development of neurons and skeletal muscle. In vitro studies have also shown similar impacts on cellular differentiation. For example, neurite outgrowth was inhibited when Neuro-2a cells were exposed to 3 μM arsenic trioxide.21 Neurogenesis and myogenesis were reduced when P19 mouse stem cells were exposed to 0.5 μM arsenite due to reductions of many cell lineage specific transcription factors (TFs), such as MyoD, myogenin, NeuroD, and neurogenin.22 Since arsenic appears to impact skeletal muscle and neurons during embryogenesis, there is likely a common progenitor cell type that arsenic is impacting. One potential target cell type is the progenitors of the neural crest (NC), termed neural plate border specifier (NPBS) cells. These cells send out signals into the neural plate border (NPB) region to induce neural crest (NC) formation and also delineate the location of the somites.23 Numerous signals, including the Wnt signaling pathway,24 are then sent into the B


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Figure 2. Arsenic decreases Sox10 intensity and MyoD nuclear localization in day 3 embryoid bodies. After exposure to 0, 0.1, and 0.5 μM arsenic for 3 days, embryoid bodies were fixed, embedded, and stained for Msx1, MyoD, Pax3, and Sox10 expression. Representative images are shown (A, n = 3 per group). Intensity values (B) and nuclear localization (C) were calculated. Statistical differences were determined by ANOVA followed by Tukey’s test (*, p < 0.05).

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at 4 °C, dehydrated in ethanol, cleared in xylene, embedded in paraffin, and then sectioned for use in immunohistochemistry. Synthesis of Monomethylarsonous Acid (AsIII(CH3)(OH)2, MMA III). Synthesis of the MMA III precursor [As(CH3)O]n was performed as reported30 with minor modifications. Arsenic trioxide (20 g, 101.1 mmol) was dissolved in aqueous sodium hydroxide solution (100 mL, 0.01 M) before adding methyl iodide (126 mL) and heating to reflux for 24 h. After cooling, ethanol (100 mL) was added to precipitate a white solid that was then filtered and dried, yielding disodium methylarsonate (16.48 g). Disodium methylarsonate was then dissolved in warm water (60 mL), and sulfur dioxide (generated in situ as described below) was bubbled through the solution for 15−20 min. The resulting solution was heated to boiling for 2 min, cooled to 0 °C, and neutralized with sodium carbonate until bubbling ceased. The neutralized solution was evaporated to dryness and was extracted with benzene. Benzene was removed in vacuo to yield [As(CH3)O]n as a white solid (7.5 g, 69.5% yield). The 1H NMR (CDCl3) spectrum was consistent with reported values,30 and the MALDI mass spectrum showed a single peak at 106.8 m/z for [As(CH3)O + H+]. The desired MMA III product was formed by dissolving [As(CH3)O]n in water.31 Synthesis of Dimethylarsonous Acid (AsIII(CH3)2OH, DMAIII). Synthesis of this compound was performed as reported32 with minor modifications. Cacodylic acid (12.5 g, 90.5 mmol) and potassium iodide (40 g, 240.9 mmol) were dissolved in water (50 mL), and sulfur dioxide (generated in situ) was bubbled through the solution for 10 min. A 1:1 solution of concentrated hydrochloric acid, and water (25 mL) was then added periodically to the reaction mixture over the course of 30 min until a yellow oil formed and elemental sulfur precipitated. The oil layer was separated, dried over CaCl2, and distilled to afford dimethylarsonous acid (16.8 g, 80% yield). The melting point of the purified compound was −35 °C, consistent with

MATERIALS AND METHODS

Caution: The following chemicals are hazardous and should be handled caref ully: sodium arsenite, monomethylarsonous acid (MMA III), and dimethylarsinous acid (DMA III). Prior to dilution, all three should be handled in a chemical f ume hood with appropriate personal protective equipment (gloves, laboratory coats, and safety goggles). P19 Cell Culture and Differentiation of Embryoid Bodies. The mouse P19 mouse embryonal carcinoma cell line (ATCC, Manassas, VA) was maintained in α-MEM containing 7.5% bovine calf serum (Hyclone, Logan, UT), 2.5% fetal bovine serum (Mediatech, Manassas, VA), 1% L-glutamine, and 1% penicillin/streptomycin (designated as growth medium) at 37 °C in a humidified incubator containing 5% CO2. The medium was changed every 48 h. To form embryoid bodies and induce differentiation, P19 cells were aggregated using the hanging drop method.28 Briefly, P19 cells were obtained from multiple T-75 flasks and trypsinized, and each flask of cells was separately suspended in differentiation medium (growth medium with 1% dimethyl sulfoxide) containing 0, 0.1, or 0.5 μM sodium arsenite at a density of 500 cells/20 μL drop. These concentrations correspond to 7.5 and 37.5 μg/L arsenic, and have been previously shown to not impact cellular proliferation or be overtly toxic to P19 cells.22,29 Each replicate contained 96 drops (n = 3 independent replicates per concentration and per day). The hanging drops were allowed to form into embryoid bodies (EBs) for 2 days, after which, each drop was transferred to a 96-well ultralow attachment plate containing 70 μL of fresh differentiation medium with 0, 0.1, or 0.5 μM arsenite. The EBs were collected for qPCR on days 3 and 5, and stored in RNALater at −80 °C. The EBs were collected at days 2, 3, 4, and 5, fixed in 10% neutral buffered formalin (NBF) overnight C


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Figure 3. Arsenic decreases NeuroD1 intensity and MyoD and NeuroD1 nuclear localization in day 4 embryoid bodies. After exposure to 0, 0.1, and 0.5 μM arsenic for 4 days, embryoid bodies were fixed, embedded, and stained for transcription factor expression. Representative images are shown (A, n = 3 per group). Intensity values (B) and nuclear localization (C) were calculated. Statistical differences were determined by ANOVA followed by Tukey’s test (*,p < 0.05). the reported value.32 The 1H NMR spectrum of DMA III in CDCl3 showed a single resonance at δ 1.97, and the MALDI mass spectrum showed a single peak at 123 m/z for [AsIII(CH3)2OH + H+]. In Situ Synthesis of SO2. Sodium metabisulfite (Na2S2O5, 10 g) was added to a two-neck, round-bottomed flask (250 mL), and concentrated sulfuric acid (100 mL) was added dropwise periodically as effervescence ceased. From this flask, a vacuum adaptor attached to tubing with a glass pipet at the end was used to bubble SO2 through the reaction solutions as described.33 Embryoid Body Exposure to MMA and DMA. To derive appropriate concentrations, dose−response experiments were carried out using varying MMA III (0−0.1 μM) and DMA III (0−20 μM) concentrations. The P19 cells were aggregated into embryoid bodies as described above and allowed to differentiate for 12 days, changing the medium every 48 h. Cells were examined visually for viability and differentiation. Concentrations of 0.01 and 0.05 μM MMA III, and 1 and 5 μM DMA III were chosen as appropriate levels that inhibited differentiation at the highest concentration without impacting proliferation or altering the total amount of cellular RNA (Figure S1, Supporting Information). P19 cells were then exposed to MMA III and DMA III for 3 or 5 days as described above, the EBs were collected, and either stored in RNALater at −80 °C for qPCR analysis or fixed in 10% NBF and then used for immunohistochemical analysis. Immunohistochemistry. The fixed and embedded EBs were cut in 5 μm sections, placed on slides, deparaffinized, and rehydrated in graded ethanol washes. Antigen retrieval was carried out with citric acid buffer (pH 6) and microwaving. Slides were blocked (1× PBS, 5%

BSA, and 0.05% Tween-20) for 1 h. Primary antibodies were incubated at a 1:200 dilution overnight at 4 °C and included Pax3 (GeneTex no. GTX100663), Sox 10 (Abnova no. H00006663-M01), NeuroD1 (Abcam no. AB60704), Msx1 (Sigma-Aldrich no. SAB2500650), MyoD (Santa Cruz no. SC304), and myogenin (Imgenex no IMG131). The secondary antibodies (1 μg/mL) conjugated to Alexa Fluor 488 (antigoat), Alexa Fluor 488 (antimouse), Alexa Fluor 594 (antirabbit), or Alexa Flour 647 (antimouse) (Invitrogen) were incubated with the slides, which were counterstained with DAPI (Invitrogen). Alexa Fluor 488 (antigoat), Alexa Fluor 594 (antirabbit), and Alexa Fluor 647 (antimouse) were multiplexed together for Msx1, MyoD, and myogenin staining. Alexa Fluor 594 (antirabbit) and Alexa Fluor 647 (antimouse) were multiplexed together for Pax3 and NeuroD1 staining. Alexa Fluor 488 (antimouse) was used for Sox10 staining. Slides were examined by conventional immunofluorescence on a Nikon Ti Eclipse Inverted Microscope. Analysis of Protein Expression. The overall expression of each transcription factor was calculated using ImageJ following the protocol developed by Arques.34 Briefly, a region of interest (ROI) was defined around each embryoid body in the blue (DAPI) channel and an integrated density value (IDV) calculated. This was repeated for each channel of interest. Ten representative nuclei covering different sizes and intensities throughout the blue channel ROI were marked, and an average IDV was calculated. The blue channel IDV was divided by the mean nucleus value, resulting in the average number of cells present in each ROI. Then, to calculate individual protein content per EB, each respective channel IDV (green, red, far red) was divided by the average D


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Figure 4. Arsenic decreases NeuroD1 intensity and nuclear localization in day 5 embryoid bodies. After exposure to 0, 0.1, and 0.5 μM arsenic for 5 days, embryoid bodies were fixed, embedded, and stained for Msx1, MyoD, Myogenin, Pax3, Sox10, and NeuroD1 expression. Representative images are shown (A, n = 3 per group). Intensity values (B) and nuclear localization (C) were calculated. Statistical differences were determined by ANOVA followed by Tukey’s test (*, p < 0.05).

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number of cells. To examine nuclear localization of specific transcription factors and colocalization of multiple transcription factors, a 50 μm × 100 μm grid was overlaid in the bottom righthand corner of each image. One hundred cells were counted per image and scored as to whether each transcription factor was localized in the nucleus of the cell and whether two or more transcription factors were colocalized together. qPCR. After 3 or 5 days of exposure to arsenite, MMA III, or DMA III, embryoid bodies were collected (n = 3 per treatment group per day), total RNA was extracted using TRI reagent (Sigma-Aldrich, St. Louis, MO), and cDNA was prepared using M-MLV Reverse Transcriptase (Promega). qPCR was conducted using RT2 SYBR green master mix (Qiagen, Valencia, CA) (primer information is provided in Table S1, Supporting Information). A 5-point standard curve was used to quantify transcript levels and assess reaction efficiency. Samples were run in triplicate, gene expression data were normalized using Gapdh as a housekeeper, and fold changes in expression were determined. Statistical Analysis. For image intensity or transcript levels, the replicates (n = 3) of each treatment, day, and transcription factor were averaged together and statistical significance determined by ANOVA followed by Tukey’s test (p ≤ 0.05). Nuclear localization and colocalization numbers were converted into percentages for each treatment, day, and transcription factor(s) (n = 3) and statistical significance determined by ANOVA followed by Tukey’s test (p ≤ 0.05).

RESULTS Time Course of Transcription Factor Expression and Nuclear Translocation in P19 Cell-Derived Embryoid Bodies. Prior to determining if arsenic or its metabolites impacted the differentiation of NPBS cells, a time-course of transcription factor expression during embryoid body formation was determined. Key protein markers of NPBS cells include Msx1 and Pax3.23,25 Along the neurogenic lineage, NPBS cells differentiate into NC progenitor cells by expressing the transcription factor Sox10. The NC progenitor cells then differentiate into NC cells, expressing the transcription factor NeuroD1 before developing into sensory neurons. Along the myogenic lineage, NPBS cells differentiate into myogenic progenitor cells, expressing the transcription factor MyoD. The myogenic progenitor cells then differentiate into myocytes, expressing myogenin, and then finally develop into myotubes.35,36 In the neurogenic lineage, Pax3, a marker of NPBS cells, and Sox10, a marker of neural crest progenitors, had steady, medium expression throughout days 2−5 (Figures 1−4). NeuroD1, a neural crest cell marker, is not expressed on days 2−3, but has high expression on day 4, and medium expression on day 5 (Figures 3−4). In the myogenic lineage Msx1, a NPBS E


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Figure 5. MMAIII reduces the expression of most transcription factors in day 3 embryoid bodies. After exposure to 0, 0.01, and 0.05 μM MMA III for 3 days, embryoid bodies were fixed, embedded, and stained for transcription factor expression. Representative images are shown in Figure S1, Supporting Information (n = 3 per group). Intensity values (A) and nuclear localization (B) were calculated. Statistical differences were determined by ANOVA followed by Tukey’s test (*, p < 0.05).

Figure 6. MMAIII decreases the expression of neurogenic transcription factors, and the nuclear localization of MyoD and NeuroD in day 5 embryoid bodies. After exposure to 0, 0.01, and 0.05 μM MMA III for 5 days, embryoid bodies were fixed, embedded, and stained for Msx1, MyoD, Myogenin, Pax3, Sox10, and NeuroD1 expression. Representative images are shown in Figure S2, Supporting Information (n = 3 per group). Intensity values (A) and nuclear localization (B) were calculated as in Figure 1. Statistical differences were determined by ANOVA followed by Tukey’s test (*, p < 0.05).

cell marker had low expression on day 2, high expression days 3−4, and medium expression on day 5, while MyoD, a myogenic progenitor marker, has medium expression throughout days 2−5 (Figures 1−4). Myogenin, which is a marker for myocytes, is not expressed on days 2−4, and has low expression on day 5 (Figure 4). When looking at the percent nuclear localization, Pax3 has at least 50% nuclear localization on all days examined (Figures 1−4). Sox10, Msx1, and MyoD are mainly found in the cytoplasm with 1−10% nuclear localization on day 2. Sox10 and Msx1 stay this way throughout days 2−5, while MyoD’s translocation to the nucleus increases on days 3−5 (Figures 1−4). NeuroD1 is not expressed until day 4, but once expressed, it is predominantly located in the nucleus on days 4 and 5, while myogenin, which is not expressed until day 5, is predominantly found in the cytoplasm (Figures 3−4). Arsenic Reduces the Differentiation of Neural Plate Border Specifier Cells. Next, embryoid bodies (EBs) were exposed to 0, 0.1, or 0.5 μM sodium arsenite and changes in transcription factor expression along the muscle and neural lineages examined. After 2 days of exposure, the 0.1 and 0.5 μM treated cells showed a significant 15−19% decrease in Pax3 intensity (Figure 1 A and B). After 3 days of arsenic exposure, Sox10 expression was also significantly decreased by 30% and 51% in the 0.1 and 0.5 μM treatments, respectively (Figure 2). After 4 and 5 days of arsenic exposure, NeuroD1 expression was decreased by 23% in the 0.5 μM treatment (Figures 3A and B

and 4A and B). Along the neurogenic pathway, it appears that arsenic is impacting differentiation starting on day 2 by reducing transcription factor expression in both neural crest progenitor cells and neural crest cells. Within the muscle lineage, only Msx1 expression was significantly decreased (34%) in the 0.5 μM treatment on day 3 (Figure 2 A and B). No other transcription factor levels were altered on any of the remaining days due to arsenic exposure. Monomethylarsonous Acid (MMA III) and Dimethylarsinous Acid (DMA III) Targets Neural Plate Border Specifier Cells. Since methylated arsenical metabolites often have higher toxicity than that of the inorganic species,12 P19 cells were exposed to MMA III and DMA III for either 3 or 5 days, and the expression of transcription factors along the muscle and neural lineages were examined. After 3 days of exposure to MMA III, Msx1, MyoD, Pax3, and Sox10 expression was significantly decreased in the 0.05 μM treatment by 22%, 11%, 7%, and 26%, respectively (Figure S2, Supporting Information, and Figure 5A). After 5 days of MMA III exposure, only the neurogenic lineage was affected with Pax3, Sox10, and NeuroD1 showing a significant reduction in expression of 10%, 11%, and 13%, respectively, in the 0.05 μM treatment (Figure S3, Supporting Information, and Figure 6A). None of the transcription factor levels were reduced after 3 days of DMA III exposure (Figure S4, Supporting Information, F


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Figure 8. DMA III decreases the expression of most transcription factors in day 5 embryoid bodies. After exposure to 0, 1, and 5 μM DMA III for 5 days, embryoid bodies were fixed, embedded, and stained for transcription factor expression. Representative images are shown in Figure S3, Supporting Information (n = 3 per group). Intensity values (A) and nuclear localization (B) were calculated, and statistical differences were determined by ANOVA followed by Tukey’s test (*, p < 0.05).

Figure 7. DMAIII decreases MyoD nuclear localization in day 3 embryoid bodies. After exposure to 0, 1, and 5 μM DMA III for 3 days, embryoid bodies were fixed, embedded, and stained for transcription factor expression. Representative images are shown in Figure S3, Supporting Information (n = 3 per group). Intensity values (A) and nuclear localization (B) were calculated, and statistical differences were determined by ANOVA followed by Tukey’s test (*, p < 0.05).

and Figure 7A). However, after 5 days of DMA III exposure, Msx1, MyoD, and myogenin were all significantly reduced by 17%, 12%, and 22%, respectively (Figure S5, Supporting Information, and Figure 8A). In the neurogenic pathway, Sox10 and NeuroD1 showed a slight, but significant decrease in the 5 μM treatment by 13% and 9% (Figure S5, Supporting Information, and Figure 8A). In the neurogenic pathway, it appears that MMA III reduces transcription factor expression on day 3, while DMA III does not reduce it until day 5 (Table 1). Arsenic, MMA III, and DMA III Reduce mRNA Levels of Myogenic and Neurogenic Transcription Factors. qPCR was used to further assess whether transcription factor expression was altered at the mRNA level. The patterns were concordant for all three arsenic species on day 3. For example, in the myogenic pathway on day 3, Msx1 was reduced by 56%, 56%, and 54% by arsenic, MMA III, and DMA III, respectively (Figure 9), although the reductions by DMA III did not reach statistical significance (p = 0.06). Similarly, in the neurogenic pathway, Pax3 expression was reduced by 79%, 84%, and 82% by arsenic, MMA III, and DMA III, respectively (Figure 9). By day 5, the early expressing transcription factor Msx1 is not differentially transcribed in the MMA III or DMA III exposures but are reduced in the arsenic-exposed cells. However, Pax3 is actually induced in the MMA III and DMA III exposure groups, while being repressed by 57% in the arsenic exposure (Figure 9).

NeuroD, a late-expressing neurogenic transcription factor, is reduced in both the arsenic and MMA III exposure groups by 50−55% (Figure 9). The late-expressing myogenic transcription factor, myogenin, is not altered. However, just like with its protein expression, the levels of myogenin transcript on day 5 are quite low. Arsenic Alters Nuclear the Localization of Transcription Factors. The cellular location of transcription factors was also examined. In the myogenic pathway after 2 days of arsenic exposure, arsenic significantly reduced the nuclear expression of Msx1 in the 0.5 μM arsenite group by 70% (Figure 1C). On days 3, 4, and 5, MyoD nuclear localization continues to be reduced by 54%, 14%, and 16%, respectively (Figures 2−4C). On days 4 and 5, NeuroD1 nuclear expression was significantly reduced in the 0.5 μM treatment by 10% and 29%, respectively (Figures 3−4C). MMA III DMA III Alters the Nuclear Localization of Transcription Factors. After 3 days of exposure to MMA III (0.05 μM) and DMA III (5 μM), MyoD nuclear localization was significantly decreased by 49% and 33%, respectively (Figures 5B and 7B, and Table 2). After 5 days of exposure to MMA III and DMA III, MyoD nuclear localization was again significantly decreased by 12% and 24% (Figures 6B and 8B). NeuroD1 nuclear localization was significantly decreased in G


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Chemical Research in Toxicology Table 1. Changes in Overall Transcription Factor Protein and mRNA Expression after Exposure to Arsenite (iAs), Monomethylarsonous Acid (MMA III), and Dimethylarsinous Acid (DMA III) during P19 Cell Differentiationa

a

An arrow indicates reduced expression, a dash indicates no change in expression, and an X indicates that the TF was not expressed on that day. The first symbol in a box represents the mRNA expression, and the second symbol represents protein expression. If the two are concordant, only one symbol is present.

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DISCUSSION The results from this study show that arsenite, monomethylarsonous acid (MMA III), and dimethylarsinous acid (DMA III) decrease the expression and nuclear localization of transcription factors involved in neurogenesis and myogenesis. These changes occur during early cell fate determination and differentiation. Interestingly, arsenical species reduce the expression of transcription factors involved in sensory neuron formation, while altering the nuclear translocation of transcription factors involved in skeletal muscle formation. P19 embryonic stem cells can be induced to differentiate into both skeletal muscles and sensory neurons by first forming embryoid bodies in the presence of DMSO.22,37 This implies that during the early stages of P19 cell differentiation, a common progenitor cell to skeletal muscle and sensory neuron is formed. During gastrulation, when the epiblast forms the three germ cell layers, some of the ectodermal precursor cells can give rise to cells in more than one germ layer, called NPBS cells.38,39 These cells express transcription factors involved in mesoderm and skeletal muscle formation, such as Pax3, Pax7, Dlx5, Msx1, and Msx2, and are responsible for secreting factors to form neural crest specifier cells, which express FoxD3, Sox9, and Sox10.40−42 Neural crest specifier cells produce neural crest cells, which develop into sensory neurons, glia, and melanocytes.43 In this study, the expression or nuclear localization of markers of NPBS cells such as Pax3 and Msx1 were decreased on day 2 of embryoid body formation. We have seen similar reductions in Pax3 protein expression in day 2 embryoid bodies exposed to 0.1 and 0.5 μM, and in Pax3 transcript expression in day 5 EBs.22 Day 2 is the earliest time point examined, as in our hands, day 1 embryoid bodies are not aggregated tightly enough and fall apart during attempts to fix and embed them. We have noted that Msx2, another marker of NPBS cells, was reduced at the transcript level (data not shown) but did not investigate its expression at the protein level. Upon the basis of protein and transcript expression of Pax3, Msx1, and Msx2, it appears that the P19 cells are differentiating into NPBS cells very early during embryoid body formation.

both the MMA III and DMA III treatments at the highest concentrations after 5 days of exposure by 30% (Figures 5C and 7C). These expression patterns are exactly the same as those of inorganic arsenic (Table 2). Arsenical Compounds Alter Co-Localization Patterns. In addition to looking at overall intensity and nuclear localization of the transcription factors, their colocalization patterns within the cells were also examined. In the myogenic pathway, Msx1 and MyoD colocalized together predominately in the cytoplasm on days 2−4. However, the frequency of them being together in the same cell drops from 98% on day 2 to 73% on day 3 and only 38% on day 4 (Figures 1−3). When treated with inorganic arsenite, on day 2 of differentiation, the overall colocalization of these two transcription factors is significantly decreased (Figure 1). By differentiation day 4, arsenic exposure does not alter the overall percentage of cells coexpressing Msx1 and MyoD; however, arsenic does significantly increase the percentage of cells expressing these transcription factors exclusively in the cytoplasm from 34% to 42% (Figure 3). In the neurogenic pathway, Pax3 and NeuroD1 are expressed in the same cells 82−84% of the time on days 4 and 5. Their coexpression is predominately located in the nuclei (Figures 3−4), although this drops from 77% on day 4 to 66% on day 5. When cells are exposed to inorganic arsenic, there is a significant shift toward the coexpression being in the cytoplasm rather than in the nuclei, such that on day 5, colocalization of Pax3 and NeuroD in the nucleus drops from 66% of cell to 48% of cells. After exposure to MMA III, similar colocalization patterns were seen in the myogenic pathway. For example, the frequency of Msx1 and MyoD being expressed in the same cell on day 3 of differentiation decreases from 78% in the controls to 66% in the 0.05 μM MMA III group (Figure 5). With DMA III, while there is not an overall reduction in Msx1 and MyoD expression in the same cell, there is a shift in cellular localization patterns. DMA III exposure reduces the nuclear coexpression of Msx1 and MyoD by 73% (Figure S4, Supporting Information). Interestingly, neither MMA III nor DMA III alter the colocalization of Pax3 and NeuroD on day 5 of differentiation (Figures S3 and S5, Supporting Information). H


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Figure 9. Arsenic, MMA III, and DMA III also alter mRNA expression of transcription factors in day 3 and 5 embryoid bodies. RNA was extracted from day 3 and 5 embryoid bodies exposed to 0, 0.5 μM arsenic, 0.01 μM MMA III, 0.05 μM MMA III, 1 μM DMA III, or 5 μM DMA III. Transcript levels of Pax3, Sox10, NeuroD1, Msx1, MyoD, and myogenin were assessed by qPCR (n = 3 per group per day). Levels were normalized to Gapdh as a housekeeping gene, and expression fold changes were compared to those of the control cells. Statistical differences were determined by ANOVA followed by Tukey’s test (*, p < 0.05).

cell marker, on days 4 and 5. Once Pax3 is bound to the DNA, it interacts with Sox10 via the Pax3 paired domain (PD)49 resulting in increased transcriptional activity of target genes. Sox10 can then activate Islet1, which then activates NeuroD.50 Normally, Sox10 expression begins when neural crest progenitors start to migrate from the neural tube, and its expression starts to decrease as the cells differentiate into neural crest cells, which express NeuroD1.51 Arsenic significantly reduces the expression of neurogenic transcription factors in a time-dependent manner, such that Pax3 protein and transcript levels are reduced on day 2, Sox10 protein and transcript levels are reduced on day 3, and NeuroD protein and transcript levels are reduced on days 4 and 5.

During embryogenesis, Pax3 expression is activated by the Wnt/β-catenin pathway.44 Pax3 then helps regulate the differentiation of neurons and skeletal muscle by activating further transcription factors required for neurogenesis and myogenesis, such as myogenin and MyoD.36,45 In addition to regulating skeletal myogenesis, Pax3 also plays a role in the formation of neural crest progenitor cells. For example, Pax3 and Zic1 together are sufficient to start neural crest development and differentiation46,47 by upregulating such genes as neurogenin 2.48 Indeed, the reduction in Pax3 transcript and protein expression may help explain the decrease in Sox10, a marker of neural crest progenitors, on day 3, and a reduction in NeuroD1, a neural crest I


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Table 2. Changes in the Nuclear Translocation of Transcription Factors after Exposure to Arsenite (iAs), Monomethylarsonous Acid (MMA III), and Dimethylarsinous Acid (DMA III)a

a

An arrow indicates reduced expression, a dash indicates no change in expression, and an X indicates that the TF was not expressed on that day.

bodies.22 Similarly, NeuroD expression is also regulated at the transcriptional level, particularly due to changes mediated by chromatin remodeling proteins.64 Although NeuroD is not expressed until later in embryoid body formation, its protein and transcript expression is also reduced in day 5 embryoid bodies. How specifically arsenic reduces transcription factor expression is not fully understood. However, many studies suggest that arsenic changes the epigenetic regulation of these genes by altering DNA methylation and histone modification patterns.65 Interestingly, distinct DNA methylation profiles in blood leukocytes are correlated with differing levels of MMA III, DMA III, and arsenic in urine,66 while p16 methylation efficiency is negatively correlated with the levels of urinary DMA III compared to MMA III or inorganic arsenic.67 These studies suggest that each arsenical species targets overlapping, yet distinct areas of the genome. Indeed, our study demonstrates that exposure to MMA III and DMA III resulted in several key differences in transcription factor expression compared to arsenite itself. MMA III reduces Pax3 and MyoD protein levels, which was not seen with arsenite (Table 1). While DMA III does not alter the expression of any of the transcription factors at day 3, it reduces all of them except Pax3 on day 5 of embryoid body formation. However, there was no difference in nuclear translocation between arsenite, MMA III, and DMA III (Table 2). These findings show that MMA is more toxic to the cells at a lower concentration than sodium arsenite, which is consistent with other studies.12,68 It is also interesting to note that P19 appears to have arsenic methylation capabilities. Arsenite methyltransferase (As3MT) is the enzyme that transfers the methyl group from SAM turning arsenite into MMA III,69,70 and at least its transcript is present in stem cells both before and after differentiation into embryoid bodies (Supporting Information, Figure 5). However, whether arsenite is being metabolized into MMA III and DMA III is unknown, but given that each of the arsenical species appears to have a slightly different impact on transcription factor expression patterns, this suggest that a portion of the arsenite remains in its inorganic form. Importantly, the results of this study demonstrate that low concentrations of MMA III inhibit sensory neuron and skeletal

In the muscle lineage, arsenite significantly decreases the expression of the NPBS cell marker Msx1 on day 3. It is interesting to note that typically the levels of myogenic transcription factors did not change due to arsenic exposure at either the protein and transcript level, rather their nuclear translocation was reduced. Both Msx1 on day 2 and MyoD on all days expressed had significantly lower levels in the nucleus in the arsenic exposed groups. Normally, all of the basic helix− loop−helix proteins (bHLH) bind to a consensus E-box sequence (CANNTG).52 MyoD is one such bHLH protein that can bind to E-boxes and increase the transcription of skeletal muscle genes.53 A common mechanism of regulating the function of MyoD is by keeping it in the cytoplasm using retention proteins. For example, Id proteins bind to bHLH family members and play a critical role by shuttling factors such as MyoD between the cytoplasm and the nucleus.54 I-mf proteins bind to MyoD family members and mask their nuclear localization signals.55 TAZ is a coactivator of MyoD and is also retained in the cytoplasm via its binding to 14−3−3.56 Thus, Id, I-mf, and TAZ proteins act as negative regulators of bHLH proteins by retaining them in the cytoplasm. In the present study, the level of MyoD in the nucleus was significantly reduced by arsenic in all time points examined, although how specifically arsenic is mediating the cytoplasmic retention is unknown. Interestingly, subfamilies of the bHLH proteins like NeuroD also recognize E-box sequences.53 Perhaps similar mechanisms are occurring with NeuroD, as its nuclear localization was also reduced in the arsenic exposed embryoid bodies. There is evidence that NeuroD also relies on heterodimerization prior to nuclear import with E4757 and that several members of the Id family (Id2 and Id4) can inhibit the activity of both MyoD and NeuroD.58,59 In addition to preventing nuclear translocation, MyoD’s ability to activate myogenic gene expression is also regulated on several other levels, including changes in DNA’s binding affinity between it and other E-box proteins, such as Twist or ZEB1,60,61 or reducing its transcription altogether by proteins such as Mist1 and MyoR.62,63 Although arsenite itself did not alter MyoD protein expression at these early time points, small reductions have been seen at the transcript level in day 5 embryoid J


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(2) Yang, Q., Jung, H. B., Culbertson, C., Marvinney, R., Loiselle, M., Locke, D., Cheek, H., Thibodeau, H., and Zheng, Y. (2009) Spatial pattern of groundwater arsenic occurrence and association with bedrock geology in greater Augusta, Maine. Environ. Sci. Technol. 43, 2714−2719. (3) Mondal, D., Banerjee, M., and Kundu, M. (2010) Comparison of drinking water, raw rice and cooking of rice as arsenic exposure routes in three contrasting areas of West Bengal, India. Environ. Geochem. Health 32, 463−477. (4) Desbarats, A. J., Koenig, C. E. M., Pal, T., Mukherjee, P. K., and Beckie, R. D. (2014) Groundwater flow dynamics and arsenic source characterization in an aquifer system of West Bengal, India. Water Resour. Res. 5, 4974−5002. (5) Zavala, Y. J., and Duxbury, J. M. (2008) Arsenic in rice: I. Estimating normal levels of total arsenic in rice grain. Environ. Sci. Technol. 42, 3856−3860. (6) Zavala, Y. J., Gerads, R., Gorleyok, H., and Duxbury, J. M. (2008) Arsenic in rice II. Arsenic speciation in USA grain and implications for human health. Environ. Sci. Technol. 42, 3861−3866. (7) Davis, M. A., Mackenzie, T. A., Cottingham, K. L., GilbertDiamond, D., Punshon, T., and Karagas, M. R. (2012) Rice consumption and urinary arsenic concentrations in U.S. children. Environ. Health Perspect. 120, 1418−1424. (8) Stýblo, M., Drobná, Z., Jaspers, I., Lin, S., and Thomas, D. J. (2002) The role of biomethylation in toxicity and carcinogenicity of arsenic: a research update. Environ. Health Perspect. 110 (Suppl 5), 767−771. (9) Concha, G., Vogler, G., Lezcano, D., Nermell, B., and Vahter, M. (1998) Exposure to inorganic arsenic metabolites during early human development. Toxicol. Sci. 44, 185−190. (10) Hopenhayn, C., Ferreccio, C., Browning, S., Huang, B., Peralta, C., Gibb, H., and Hertz-Picciotto, I. (2003) Arsenic exposure from drinking water and birth weight. Epidemiology 14, 593−602. (11) Gardner, R. M., Nermell, B., Kippler, M., Grandér, M., Li, L., Ekström, E. C., Rahman, A., Lönnerdal, B., Hoque, A. M., and Vahter, M. (2011) Arsenic methylation efficiency increases during the first trimester of pregnancy independent of folate status. Reprod. Toxicol. 31, 210−218. (12) Wang, Q. Q., Lan, Y. F., Rehman, K., Jiang, Y. H., Maimaitiyiming, Y., Zhu, D. Y., and Naranmandura, H. (2015) Effect of arsenic compounds on the in vitro differentiation of mouse embryonic stem cells into cardiomyocytes. Chem. Res. Toxicol. 28, 351−353. (13) Raqib, R., Ahmed, S., Sultana, R., Wagatsuma, Y., Mondal, D., Hoque, A. M., Nermell, B., Yunus, M., Roy, S., Persson, L. A., Arifeen, S. E., Moore, S., and Vahter, M. (2009) Effects of in utero arsenic exposure on child immunity and morbidity in rural Bangladesh. Toxicol. Lett. 185, 197−202. (14) Ogata, T., Nakamura, Y., Endo, G., Hayashi, T., and Honda, Y. (2014) Subjective symptoms and miscarriage after drinking well water exposed to diphenlyarsinic acid. Nihon Koshu Eisei Zasshi 61, 556−564. (15) Jin, Y., Xi, S., Li, X., Lu, C., Li, G., Xu, Y., Qu, C., Niu, Y., and Sun, G. (2006) Arsenic speciation transported through the placenta from mother mice to their newborn pups. Environ. Res. 101, 349−355. (16) Tsai, S. Y., Chou, H. Y., The, H. W., Chen, C. M., and Chen, C. J. (2003) The effects of chronic arsenic exposure from drinking water on the neurobehavioral development in adolescence. Neurotoxicology 24, 747−753. (17) Wasserman, G. A., Liu, X., Parvez, F., Ahsan, H., Factor-Litvak, P., van Geen, A., Slavkovich, V., Loiacono, N. J., Cheng, Z., Hussain, I., Momotaj, H., and Graziano, J. H. (2004) Water arsenic exposure and children’s intellectual function in Araihazar, Bangladesh. Environ. Health Perspect. 112, 1329−1333. (18) Dakeishi, M., Murata, K., and Grandjean, P. (2006) Long-term consequences of arsenic poisoning during infancy due to contaminated milk powder. Environ. Health 5, 31. (19) Wasserman, G. A., Liu, X., Parvez, F., Ahsan, H., Factor-Litvak, P., Kline, J., van Geen, A., Slavkovich, V., Loiacono, N. J., Levy, D., Cheng, Z., and Graziano, J. H. (2007) Water arsenic exposure and

muscle formation. The concentrations exposed to P19 cells were 10-fold lower than that of arsenite itself, resulting in a concentration of 3.8 μg/L MMA III. This is significant because it is 2-fold lower than reported levels of 7.5 μg/L MMA III in cord blood from pregnant women drinking water containing 90 ppb arsenic.71 Although speciation of the arsenical compounds was not done, a recent study investigating the general Flemish population demonstrated that an infant’s birth weight was reduced by 47 g for each 1 μg/L increase of arsenic in their cord blood.72 Similarly, in a study on the general population in Dalian, China, infants born to mothers with arsenic blood levels greater than 5.3 μg/L weighed 220 g less than those infants whose mothers had lower blood arsenic levels.73 This study also demonstrated a significant, strong correlation between maternal arsenic and cord blood arsenic. These studies indicate that low levels of arsenic can have direct impacts on the growth of newborns. Given that our results indicate that MMA III levels of 3.8 μg/L significantly reduce sensory neuron and skeletal muscle formation and differentiation, this may provide a mechanism for the low birth weight and changes in neurological function seen in epidemiological studies.74

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ASSOCIATED CONTENT

S Supporting Information *

qPCR primer sequences; MMA III and DMA III do not alter growth of P19 cell-derived embryoid bodies after 5 or 9 days (A) nor do they alter overall RNA concentrations after 5 days of exposure (B); MMA III decreases Msx1, MyoD, Pax3, and Sox10 intensity in day 3 embryoid bodies; MMA III decreases Pax3, Sox10, and NeuroD1 intensity in day 5 embryoid bodies; DMA III caused no marked decrease in transcription factor intensity in day 3 embryoid bodies; DMA III decreases Msx1, MyoD, myogein, Sox10, and NeuroD1 intensity in day 5 embryoid bodies; differentiating P19 cells have robust expression of arsenic methyltransferase (As3MT) mRNA. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.5b00036.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 1-864-656-5050. E-mail: [email protected]. Funding

Funding for this study was provided by NIH (ES023065). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Clemson Light Imaging Facility and the Clemson Histology Core Facility for imaging and sample preparation. ABBREVIATIONS MMA III, monomethylarsonous acid; DMA III, dimethylarsinous acid; NPBS, neural plate border specifier; NPB, neural plate border; NC, neural crest; EMT, epithelial to mesenchyme transition; EB, embryoid body; PBS, phosphate buffered saline; BSA, bovine serum albumin; MEM, modified Eagle’s medium; IDV, integrated density value; bHLH, basic helix−loop−helix; As3MT, arsenic methyltransferase

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