ToxSci Advance Access originally published online on September 19, 2006
Toxicological Sciences 2006 94(2):310-321; doi:10.1093/toxsci/kfl114
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Nonylphenol and Octylphenol-Induced Apoptosis in Human Embryonic Stem Cells Is Related to Fas-Fas Ligand Pathway
* Laboratory of Stem Cell Biology, Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea
Institute of Life Science and Natural Resources, Korea University, Seoul 136-713, Republic of Korea
Laboratory of Reproductive Endocrinology, Department of Life Sciences, College of Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea
1 To whom correspondence should be addressed at College of Life and Environmental Sciences, Science Campus, Korea University, 1, Anam-dong 5-ga, Sungbuk-goo, Seoul 136-713, Korea. Fax: +82-2-3290-3507. E-mail: jhkim{at}korea.ac.kr.
Received July 26, 2006; accepted September 18, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human embryonic stem (hES) cells have been proposed as a source of various cell types for cell replacement therapy. Besides their potential in therapeutic uses, ES cells also have other potential applications, such as in drug discovery and in vitro screening assays of various toxicants. Nonylphenol (NP) and octylphenol (OP) are common environmental contaminants, known to disrupt the reproductive and endocrine system. However, little is known about their toxicological effects on early embryonic development in humans. In this study, we used undifferentiated hES cells and the neural progenitor cells derived from them to investigate the potential toxicity of NP and OP. Our results show that the cytotoxic effects of NP and OP involve DNA fragmentation, the major characteristic of apoptosis. The NP- and OP-induced apoptosis was concomitant with the increased activity of Caspase-8 and -3. Moreover, both Fas and Fas ligand (FasL) protein expressions were markedly increased in the NP- or OP-exposed hES cells. These results suggest that NP and OP are able to trigger apoptosis in hES cells via a pathway dependent on caspase activation and Fas-FasL interaction. In particular, hES cell–derived neural progenitor cells had a higher sensitivity to the toxicants than undifferentiated hES cells, thereby suggesting that the toxic stress response may differ depending on the developmental stage. These findings offer new perspectives for understanding the fundamental mechanisms in chemical-induced apoptosis in hES cells.
Key Words: nonylphenol; octylphenol; human embryonic stem cell; apoptosis; Fas-Fas ligand pathway; caspase.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human embryonic stem (hES) cells have been suggested to be a useful source for cell therapy because they have the capacity to self-renew and the ability to generate a wide range of cell types. In addition to its potential in the therapeutic uses, hES cells are also expected to provide a new platform for testing the possible toxic effects of new drugs, chemicals, or environmental contaminants (Davila et al., 2004
; Gorba and Allsopp, 2003; McNeish, 2004). To date, most information on the action mechanisms of various toxicants has been derived from animal testing or in vitro studies using highly differentiated, immortalized, or tumoriogenic cells. It was recently suggested that sensitivity to hazardous effects of the toxicants may differ according to the developmental and differentiational state of the target cells and tissues (Kudo et al., 2004). Therefore, new toxicological approaches are required to assess their effects on the cellular developmental processes and gene expression patterns of early embryogenesis. In this respect, ES cells may offer considerable advantages in that they are genetically normal, demonstrate uniform physiological responses to growth factors and a variety of other extracellular signals, and are grown at a scale suitable for use in high-throughput screening (Keller, 2005; McNeish, 2004). In particular, the use of hES cells for toxicological applications can provide an understanding of the effects of various chemicals on early human embryonic development and contribute to the identification of clinically relevant biomarkers. Although the recent establishment of hES cells has opened the possibilities for these studies (Thomson et al., 1998), there are few reports on the cytochemical characterizations of hES cells that can be used to monitor the harmful effects of various toxicants.
Nonylphenol (NP) and octylphenol (OP) are ubiquitous pollutants in the environment. Due to their widespread uses in many industrial and agricultural products, it is believed that high amounts of NP and OP have been discharged to aquatic ecosystems (Ying et al., 2002). Moreover, these chemicals can accumulate within the internal organs of fish and birds, reaching a concentration 10–100 times higher than that found in the environment based on their high stability and lipid solubility. These substances are generating considerable concern within the public and scientific community because they can easily pass to humans through the food chain and mimic the actions of natural hormones in the body. Recent studies reported that the levels of these chemicals present in the environment may be sufficient to disrupt the reproductive and endocrine system (Chapin et al., 1999; Ying et al., 2002). All evidence reported in recent years has emphasized the need for more effective and reliable methods for the risk assessment of these substances in humans. However, a number of previous studies have focused on the effects of toxicants on the relatively late developmental process including fetal or neonatal stage of animal models (Rankouhi et al., 2004; Sweeney, 2002; Wang et al., 2003), and little is known about their effects on the early embryonic development, particularly in humans.
Although the cytotoxicity of NP and OP is well-known, the effects of these chemicals on cell death and the related underlying mechanisms are not fully understood. Moreover, to our knowledge, the mechanism of apoptosis induced by environmental contaminants in hES cells has not been demonstrated previously. Accordingly, we investigated the ability of NP and OP to induce apoptosis in hES cells and examined their signaling pathway. In particular, this study focused on the death receptor pathway by determining the changes in Fas and Fas ligand (FasL) expressions as well as the activation of the related caspases in this pathway. We also compared the effects of these chemicals on undifferentiated hES cells and the neural progenitor cells differentiated from them.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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hES cell culture and neural differentiation.
hES cell lines, Miz-hES4 and Miz-hES6 (Kim et al., 2005
; Son et al., 2005; Yoo et al., 2005), were cultured on mitomycin C–treated mouse embryonic fibroblast (MEF) feeder cells in Dulbecco's modified Eagle's medium/F12 medium (Gibco BRL, Grand Island, NY) supplemented with 20% KnockOut SR (Gibco BRL), 0.1mM nonessential amino acids (Gibco BRL), 0.1mM ß-mercaptoethanol (Sigma Chemical, St Louis, MO), 100 unit/ml penicillin/streptomycin (Gibco BRL), and 4 ng/ml human basic fibroblast growth factor (hbFGF, R&D Systems, Minneapolis, MN). Cultures of hES cells were routinely passaged at approximately weekly intervals by dissociation with 1 mg/ml collagenase type IV (Gibco BRL). For chemical exposure, the hES cells were cultured in the absence of the feeder layer on 20 µg/ml laminin-coated plates in MEF-conditioned medium (MEF-CM) as previously described (Xu et al., 2001). In contrast to their murine counterparts, hES cells grow very poorly when dissociated to single cells. Therefore, in the present study, the dissociated hES cell colonies were suspended in the MEF-CM, gently triturated to make small clusters, and then, a portion of the suspension was trypsinized for counting the cell number. The rest of the untrypsinized suspension was used for cell plating, based on the counts of the trypsinized suspension. The MEF-CM was supplemented with 5 ng/ml of hbFGF before feeding the hES cells.
To generate neural progenitor cells, hES cells were differentiated as previously described (Kim et al., 2003). Briefly, the hES colonies were harvested upon treatment with collagenase type IV (Invitrogen, Grand Island, NY), gently triturated, and cultured for 8 days in suspension culture dishes (Corning, Cambridge, MA) containing hES cell culture medium without hbFGF to form embryoid bodies (EBs). The EBs were then plated onto tissue culture dishes in insulin/transferrin/selenium/fibronectin (ITSFn) medium (Kim et al., 2003). Over the span of 8 days, neural progenitor cells were observed to migrate from the EBs. The neural progenitor cells were dissociated with 0.05% trypsin (Invitrogen) and plated on poly-L-ornithine (15 g/ml, Sigma)–coated plates at a density of 2 x 105 cells/cm2 in N2 medium (Kim et al., 2003) containing hbFGF (20 ng/ml). The differentiation of neural progenitor cells from hES cells was confirmed by the typical bipolar morphology and immunostaining with an antibody against Nestin, a marker of neural progenitors. All experimental procedures involving hES cells were approved by the Ministry of Health & Welfare and Korean Stem Cell Research Center (IRB number, 24).
Exposure to NP and OP.
4-Nonylphenol (NP) and 4-tert-octylphenol (OP) were purchased from Fluka Chemica (Buchs, Switzerland; > 90% pure). Actinomycin-D (Act-D; Sigma Chemical) was used as a positive control for induction of apoptosis (Kleeff et al., 2000). Stock solutions of these compounds were prepared in dimethylsulfoxide (DMSO). The final concentration of DMSO in the culture medium was below 0.01%. At this concentration, DMSO did not affect the normal cell growth. Exposure to NP, OP, Act-D, or vehicle (DMSO) was initiated after 48 h of plating the undifferentiated hES cells without a feeder layer or after 4 days of culturing the hES cell–derived neural progenitor cells in N2 medium when cells become 70–75% confluent after attachment. Twenty-four to forty-eight hours after the initial exposure, the undifferentiated hES cells and the neural progenitor cells were examined morphologically and by other methods described below. A dose range of 12.5–200µM was tested using cytotoxicity assays. These doses were chosen based on previous studies (Hughes et al., 2000; Raychoudhury et al., 1999). For evaluation of apoptosis, a dose range of NP and OP was decided according to the data obtained from the cytotoxicity assay.
Cytotoxicity assays.
To assess cell viability and cytotoxicity, cells exposed to NP or OP were stained with acetoxymethyl diacetylester of calcein (calcein-AM; Molecular Probe, Eugene, OR) and propidium iodide (PI; Sigma Chemical). After NP or OP exposure, the cells were harvested with trypsin/EDTA (Invitrogen), washed in phosphate-buffered saline (PBS), and incubated with 50µM calcein-AM at 37°C for 45 min followed by 10 µg/ml PI for 10 min. Because ES cells are known to express multidrug resistance proteins that may pump out calcein-AM, the hES cells were exposed to a relatively high amount of calcein-AM (50µM) and the green fluorescence of its byproduct, calcein was examined immediately under fluorescence microscopy (Axiocam, Zeiss, Germany). Cell viability was also quantitatively determined by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) assay. The hES cells were plated onto 48-well plates at a density of 5 x 104 cells per well. Different concentrations of NP or OP were then added to the wells. After 24 or 48 h of incubation, 0.1 mg/ml MTT in ES medium was added to the cells and incubated for 4 h. The resulting formazan crystals were dissolved in the DMSO. The absorbance was measured using spectrophotometer (Libra S22, Biochrom, Cambridge, UK) at a wavelength of 570 nm.
In situ detection of apoptosis.
Apoptosis was detected using the terminal deoxynucleotidyl transferase–mediated d-UTP nick end-labeling (TUNEL) method. The cells were fixed in 4% paraformaldehyde (PFA), pH 7.4 for 20 min at 4°C. After permeabilization with 0.1% Triton X-100 in 0.1% sodium citrate, TUNEL was performed using an in situ Cell Death Detection Kit (Roche, Penzberg, Germany) according to the manufacturer's instructions. The cells were counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). The numbers of TUNEL-positive cells were counted, scoring at least 900 cells in five microscopic fields randomly selected in each group.
Quantification of hypodiploid DNA content by flow cytometry.
After exposure to NP or OP for 48 h, the cells were harvested and fixed overnight with 80% ethanol at – 20°C. The cells were then centrifuged at 2000 x g at 4°C, washed with PBS, and incubated with PBS containing 10 µg RNase A for 45 min at 37°C. After incubation, 10 µg PI was added, and the cells were further incubated for 30 min. Detection of hypodiploid DNA was analyzed by determining the DNA content in the nuclei by flow cytometry. The assessed histograms were generated using the computer program Modfit LT 3.1 (Verity Software House, Topsham, ME).
Semiquantitative reverse transcriptase–polymerase chain reaction.
Total RNA was isolated from the cells using the TRIzol (Invitrogen) method. Complementary DNA was synthesized from 1 µg of the total RNA using a RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, Hanover, MD) according to the manufacturer's protocol. The cDNA obtained was used as a template for subsequent PCR amplification using an AccuPower PCR Premix (Bioneer, Daejeon, Korea). The human-specific primer sequences and PCR conditions used in this study are summarized in Table 1. Under these conditions, preliminary experiments demonstrated that the plateaus for amplification had not been reached. Analysis of the respective bands was performed using an image analyzer (AutoChemi Bioimaging system, UVP, Inc., Upland, CA). The levels of target mRNA expression were normalized based on the signal from glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA. There were no noticeable effects on GAPDH expressions by NP and OP exposure.
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Determination of active Caspase-8 and -3.
The potential role of the Caspase-8 and -3 proteases in the pathway of NP- and OP-induced apoptosis was examined by detecting the active forms of each caspase using the specific antibody for active Caspase-3 and a CaspaTag Caspase-8 In Situ Assay Kit (all from Chemicon International, Inc., Temecula, CA) according to the manufacturer's protocol.
Immunocytochemistry.
For immunostaining of Oct4, Nanog, Fas, FasL, and Nestin, the cells were grown on 4-well or 24-well plates and exposed to the chemicals according to the methods described above. The cells were fixed in cold 4% PFA for 20 min at 4°C. After blocking and permeabilization with 0.3% Triton X-100 and 10% goat serum in 0.1% BSA/PBS, the cells were probed with primary antibodies to Oct4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), Nanog (R&D Systems), Fas (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), FasL (PharMingen, San Diego, CA), FasL (Santa Cruz Biotechnology, Inc.), and Nestin (R&D Systems). After incubation with the primary antibodies, the cells were washed and then probed with appropriate Alexafluor secondary antibodies (Molecular Probe) for 90 min in the dark at room temperature. Finally, the fluorescent images were captured using a fluorescence microscope (Axiocam) after counterstaining with DAPI.
Western blot analysis.
The lysates from cells were run on 4–12% gradient polyacrylamide gels and transferred to nitrocellulose filters (all from Invitrogen). The filters were blocked for 1 h at room temperature in 5% skim milk in TBS-T (10mM Tris-HCl pH 7.9, 150mM NaCl, 0.05% Tween-20) and incubated overnight at 4°C with primary antibodies to Oct4 (Santa Cruz Biotechnology, Inc.), Nanog (R&D Systems), Fas (Santa Cruz Biotechnology, Inc.), FasL (PharMingen), FasL (Santa Cruz Biotechnology, Inc.), or active Caspase-3 (Chemicon International, Inc.). The filters were then washed in TBS-T, and the appropriate horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology) were added and probed for 1 h at room temperature. The filters were washed in TBS-T, and the signals were detected by chemiluminescence using ECL reagents (Santa Cruz Biotechnology, Inc.).
Statistical analysis.
Results shown are the average of three independent experiments (n = 3). The values are expressed as means ± SE. For the determination of statistical significance, results were analyzed by a one-way ANOVA, followed by the Bonferroni's test procedure for multiple comparisons with the appropriate control. p Values less than 0.05 were judged to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effects of NP and OP on Cell Viability and Cytotoxicity in hES Cells
To examine the effects of NP and OP on cell viability and cytotoxicity, hES cells were exposed to NP or OP in feeder-free conditions for 24 or 48 h and stained with calcein-AM and PI. Dead cells were identified by a red fluorescence generated by PI. On the other hand, viable cells were identified by a green fluorescence generated by the enzymatic hydrolysis of calcein-AM, which only occurs in living cells as a result of esterase activity. As shown in Figure 1A, the number of PI-positive and calcein-negative cells was dose dependently increased after 48 h of NP or OP exposure, while the PI-positive dead cells were rarely found in the vehicle (DMSO)-exposed control (CV). As expected, exposure of hES cells to Act-D induced considerable cell death (Fig. 1A).
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The viability of hES cells after exposure to NP, OP, or Act-D was also determined quantitatively by the extent of cellular metabolism with an MTT assay. After 24 h of exposure, hES cell survival was only significantly affected by exposure to 200µM of NP (Fig. 1B). However, a more significant decrease in viability was observed after 48 h of exposure. Compared to the vehicle-exposed cells, there was an approximately 30% decrease after 100µM of NP or OP exposure and a more than an 80% decrease after 200µM of NP or OP exposure (Fig. 1B).
Effects of NP and OP on the Pluripotent Status of hES Cells
To test whether hES cells maintain their undifferentiated state under NP or OP exposure, the expressions of transcription factors associated with pluripotent stem cells were evaluated at the mRNA and protein levels. The expressions of Oct4, Rex1, and Nanog mRNAs were not altered after exposure to NP or OP except for 10µM of Act-D, which caused a significant decrease in the expression of Nanog gene (Fig. 2A). However, the expressions of Oct4 and Nanog proteins were dose dependently decreased after the NP or OP exposure (Fig. 2B). In particular, 100µM of NP or OP markedly reduced the expression levels of these proteins to the levels similar to that decreased by Act-D (Fig. 2B). The cellular localizations and expression patterns of Oct4 and Nanog were also evaluated by immunocytochemistry. The number of cells expressing both Oct4 and Nanog was markedly decreased, and condensated or fragmented nuclei were often found in the NP- or OP-exposed cells (Fig. 2C, arrows). We also found that some of the cells exposed to NP or OP expressed neither of Oct4 and Nanog (Fig. 2C, dotted circles).
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The decreased expressions of Oct4 and Nanog were concomitant with the altered cellular morphologies. The phase-contrast observation of hES cells exposed to vehicle alone showed the typical morphology of undifferentiated hES cells (prominent nucleoli and high nuclear/cytoplasmic ratio), and detachment of cells was rarely monitored (Fig. 2C, top left panel). In contrast, the cells exposed to NP or OP showed differentiated morphologies (elongated cells with extended cytoplasm), and there was a dose-dependent detachment of cells (Fig. 2C, left panels).
Analysis of DNA Fragmentation and Hypodiploid Cells after NP and OP Exposure
As nuclear staining with DAPI revealed an increased number of condensated or fragmented nuclei when hES cells were exposed to NP or OP (Fig. 2C), we next investigated whether NP or OP induces apoptosis in hES cells by using in situ TUNEL analysis. Similar to Act-D, NP or OP exposure significantly increased the percentages of TUNEL-positive cells (26.3 ± 4.1% and 22.8 ± 1.2%, respectively) as compared to the control values (3.4 ± 0.6 %) (Figs. 3A–F).
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To further confirm the chemical-induced apoptosis, hES cells were stained with PI and analyzed for hypodiploid cells by flow cytometry. The fraction of apoptotic cells was identified as a sub-G1 hypodiploid population in a DNA histogram. As shown in Figure 3G, the hES cells exposed to NP for 24 h had the sub-G1 hypodiploid peak, and the percentage of cells with fragmented DNA was much higher than the CV. Comparable results were obtained from the OP exposure (data not shown). These observations thus confirm that NP and OP are able to trigger apoptosis in hES cells.
Activation of Caspase-3 and -8 in hES Cells Undergoing Apoptosis
To elucidate the molecular pathways involved in the NP- and OP-induced apoptosis, expressions of Caspase-3 and -8 were measured in the hES cells following NP or OP exposure. Among the various caspases, Caspase-3 is considered to be a major executioner of apoptosis because it generally participates in all major apoptotic signal transduction pathways. No significant change in the mRNA expression of Caspase-3 was observed in the hES cells exposed to NP or OP, compared to the vehicle-exposed cells (Fig. 4A). Considering that the cleavage of Caspase-3 is induced at the onset of apoptosis, we next examined the accumulation of cleaved Caspase-3 in the NP- or OP-exposed hES cells using an antibody against the active fragment of Caspase-3, p17. Western blot analysis showed that the accumulation of cleaved Caspase-3 was increased in the hES cells exposed to 5µM of Act-D or 100µM of NP or OP for 24 h compared with the CV, while exposure to DEVE-CHO, the Caspase-3 (CPP32)–specific inhibitor, prior to NP exposure, inhibited the accumulation of cleaved Caspase-3 (Fig. 4B). The activation of Caspase-3 was further supported by immunostaining analysis. Almost all of the hES cells showing fragmented nuclei were positive for the active form of Caspase-3, and exposure to NP or OP increased the amount of its active form compared to the vehicle-exposed cells (Fig. 4C, upper panels).
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Caspase-8 is one of the most upstream protease of the activation cascade of caspase, and the activation of Caspase-8 induces the proteolytic cleavage of Caspase-3, leading to activation of Caspase-3. To investigate the activation of Casepase-8 under NP or OP exposure, we used the fluorogenic inhibitor (FAM-LETD-FMK) which can specifically bind to the active Caspase-8. Compared with CV, an increase in Caspase-8 activation was detected in the hES cells exposed to Act-D or 100µM of NP or OP (Fig. 4C, lower panels), and the activation of Caspase-8 occurred concomitantly with Caspase-3 activation in some of the cells (Fig. 4C, insets in lower panels). Although Caspase-8 activation was detected in most of the cells positive for active Caspase-3, not all of the cells showing Caspase-3 activation displayed Caspase-8 activity.
Changes on the Fas and FasL Expressions after NP or OP Exposure
To further investigate whether NP- or OP-induced apoptosis occurred via Fas and FasL interaction, which is known to mediate the Caspase-8–dependent apoptotic pathway, we examined the changes on the Fas and FasL expressions in hES cells after NP or OP exposure. Western blot analysis demonstrated that the overall expression level of FasL was higher than that of Fas in hES cells, and the expressions of these proteins were increased after exposure to Act-D, NP, or OP (Figs. 5A and 5B). We used two different antibodies for FasL and these antibodies showed similar patterns. To further strengthen these findings, immunostaining of these proteins was performed. Consistent with the Western blotting results, considerable upregulation of both Fas and FasL was observed in the hES cells exposed to Act-D, NP, or OP (Fig. 5C), and there was prominent colocalization of these two proteins (Fig. 5C, insets in lower panels).
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Effects of NP and OP on Neural Progenitor Cells Differentiated from hES Cells
To investigate potential differences in sensitivity to NP or OP depending on the differentiation status, we differentiated the hES cells into neural progenitor cells and exposed them to NP or OP. Almost all the differentiated cells were identified as neural progenitors by staining with an antibody against Nestin, a marker of neuroepithelial cells. In situ TUNEL analysis showed that the number of apoptotic cells was markedly increased after 12.5µM of NP or OP exposure (Fig. 6A). Compared with the undifferentiated hES cells, the hES cell–derived neural progenitor cells were more sensitive to NP and OP. As shown in Figure 6B, much higher percentages of the TUNEL-positive cells were observed in the neural progenitor cells (22.9 ± 3.7% and 44.2 ± 1.4%) than in the undifferentiated hES cells (6.1 ± 2.2% and 7.1 ± 2.2%) after exposure to 12.5µM of NP or OP.
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The difference in sensitivity to toxicants is possibly due to the potential difference in the expression level of efflux transporters which may pump out the toxicants. To address this, we examined the changes in gene expressions of several ATP-binding cassette (ABC) transporters, which are known to be involved in drug resistance, in different stages of the neural differentiation. The differentiation of hES cells into neural progenitors was confirmed by the downregulation of Nanog, a marker of undifferentiated ES cells and the concomitant upregulation of Sox1, indicative of neural differentiation (Fig. 6C). Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of various ABC transporter genes showed that with the exception of the ABCC1/MRP1 gene, the ABCB1/MDR1, ABCC2/MRP2, and ABCG2/BCRP genes were all significantly downregulated to varying degrees upon the differentiation of hES cells into neural progenitor cells (Figs. 6C and 6D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study was designed to investigate the ability of NP and OP to induce apoptosis in undifferentiated hES cells and the neural progenitor cells derived from hES cells. By exploring its signaling pathway, we attempted to characterize the molecular and cellular nature of hES cells in response to the toxicants. Previous studies have shown that NP and OP enhance apoptosis in various cell types, such as thymocytes, PC12 cells, spermatogenic cells, and Sertoli cells (Aoki et al., 2004
; Raychoudhury et al., 1999; Wang et al., 2003; Yao et al., 2005). Recently, Kudo et al. (2004) suggested a good in vitro model system for evaluating the effect of NP on the development of central nervous system using mouse neural stem cells. Here, we extended these findings by showing that NP and OP are able to trigger apoptosis both in undifferentiated hES cells and neural progenitors derived form hES cells and that this occurs via a pathway dependent on caspase activation and Fas-FasL interaction.
Activation of the caspase cascades has been demonstrated to be essential in the induction of apoptosis in many different cell types (Kudo et al., 2004; Thorburn, 2004; Yao et al., 2005). Our results showed that NP and OP did not change the mRNA expression of Caspase-3; however, they induced the activation of Caspase-8 and -3, which led to increased apoptosis in hES cells. This result is in agreement with the study of Kudo et al. (2004), which demonstrated that NP induced apoptosis in mouse neural stem cells through the activation of Caspase-3.
Our studies focused on the involvement of the Fas and FasL pathway in NP- and OP-induced apoptosis by revealing that the expressions of Fas and FasL were upregulated in association with the activation of downstream Caspase-8 in the hES cells exposed to NP or OP. It may be worth to note that the overall expression level of FasL in hES cells was much higher than that of Fas. Although we cannot exclude the possibility that the differential expressions may be due to the 16 differences in the affinities between Fas and FasL antibodies, Fabricius et al. (2005) also demonstrated that FasL is highly expressed in mouse ES cells. They suggested that the high expression level of FasL in ES cells may induce apoptosis of T cells, thereby protecting ES cells from the immune system upon transplantation. Caspase-8, which binds Fas-associated death domain, is activated by self-cleavage as a result of oligomerization and, subsequently, activates effector caspases such as Caspase-3 (Thorburn, 2004). In this study, we showed that the protein expressions of Fas and FasL were markedly increased in the hES cells exposed to NP or OP. We also found that the increased apoptosis was accompanied by the activation of Caspase-8 and -3 and the upregulation of Fas and FasL. Although the involvement of the Fas and FasL pathway in the NP-induced apoptosis has also been reported in thymocytes and rat testes (Han et al., 2004; Yao et al., 2005), the exact mechanism underlying the inductive effect of NP and OP on the Fas and FasL pathway remains unclear. It has been shown that the stress-induced expressions of Fas and FasL are dependent on the activation of various signaling molecules such as p53, nuclear factor-kappa B, specificity protein-1, and early growth response factors (Nagata et al., 1999). Therefore, further investigations are needed to determine whether these signaling molecules are involved in the NP- and OP-induced Fas and FasL pathway.
In our study, we could not exclude the possibility that the NP- and OP-induced apoptosis was mediated by other pathways in addition to the Fas and FasL pathway. Of note, the number of NP- or OP-exposed hES cells that stained for active Caspase-3 was much higher than those that stained for active Caspase-8. This suggests that there can be some caspases other than Caspase-8 that activate the Caspase-3 in our system. There is increasing evidence that apoptosis can also be induced by a variety of signal transduction events leading to the stimulation of calcium flux, cAMP production, phopholipase C activation, inositol phosphate generation, and the mitochondrial pathway. The extent to which each pathway contributes to apoptosis in the hES cells induced by NP and OP remains to be determined.
Our data show that exposure to NP or OP altered the pluripotent state of hES cells, causing decreases in the expressions of the pluripotent markers along with changes in the cellular morphology. In addition to the toxic effects, NP and OP have been shown to have estrogenicities (Paris et al., 2002; White et al., 1994). Despite important roles of estrogen in many physiological processes, its potential effects on early embryonic development or differentiation of hES cells have not been examined extensively. It has been shown that the estrogen receptor (ER) 
and ß are found in the early developing mouse embryos (Hiroi et al., 1999). More recently, Hong et al. (2004) reported the expression of ER and ß in undifferentiated hES cells and differentiating EBs and showed that exposure of EBs to estrogen resulted in increased expressions of endodermal cell markers such as GATA-4 and -fetoprotein. These findings suggest that estrogen may play a key role in controlling the differentiation of hES cells. Since NP and OP mimic the action of estrogen by binding to ERs, it may be worthwhile to further evaluate the effects of NP and OP on the pluripotency and differentiation of hES cells.
This study was also extended to compare the effects of these chemicals between undifferentiated hES cells and neural progenitor cells, which are differentiated from hES cells. Interestingly, the neural progenitor cells were more sensitive to the cytotoxic effects of NP and OP than the undifferentiated hES cells. Several previous studies have reported that a characteristic expression of genes ("stemness" genes) in embryonic and adult stem cells provides a high resistance to stress with upregulated DNA repair, protein folding, ubiquitin systems, and detoxifier systems, compared to differentiated cells (Ramalho-Santos et al., 2002; Saretzki et al., 2004). Particularly, multidrug resistance 1 (MDR1, also known as P-glycoprotein), which belongs to the ABC transporter family, are encoded by ABCB1, one of the stemness genes related with the toxic stress response. Recent studies have provided evidence that ABCG2/BCRP and ABCB1/MDR1 are expressed in ES cells and primitive stem cells of different tissues including murine hematopoietic stem cells and human neural stem cells (Islam et al., 2005a; Islam et al., 2005b; Ramalho-Santos et al., 2002; Saretzki et al., 2004; Zhou et al., 2001). Our results show that several candidate genes of ABC transporters, such as ABCB1/MDR1, ABCC2/MRP2, and ABCG2/BCRP, are expressed in hES cells, but their expressions are downregulated during the differentiation of ES cells into EBs and the neural progenitors. Although the physiological role of ABC transporters in stem cells is unclear, they are generally known to modulate the absorption, distribution, metabolism, secretion, and toxicity of various xenobiotics (Chang, 2003; Leslie et al., 2005). Based on these previous investigations, we speculate that the more efficient stress defense in undifferentiated hES cells compared with the hES cell–derived neural progenitor cells may originate from the differences in the expression levels of the ABC transporters. Although our data clearly show that several ABC transporters are dramatically downregulated upon the neural differentiation of hES cells, we cannot rule out the possibility that other defense mechanisms may be involved in the protection of hES cells from NP or OP. Indeed, several antioxidant and stress-resistance genes are highly expressed in ES cells but become downregulated during differentiation (Ramalho-Santos et al., 2002; Saretzki et al., 2004). In addition, a previous report suggested that NP is unlikely to be a substrate of human P-glycoprotein (MDR1), one of the ABC transporters which are examined in our study. Therefore, further studies are needed to identify the NP- or OP-specific transporter and to elucidate the exact mechanism of the toxic stress defense in ES cells.
Because the existing toxicity assays using fully differentiated cell types or immortal cell lines cannot reflect a series of stages during the embryonic development, stem cells may be a solution for the analysis of developmental toxicity. In particular, hES cells can be differentiated into a variety of organ-specific stem cells and also can generate a large number of human somatic cell types. In this context of unique properties of hES cells, the development of efficient differentiation systems for generating a large number of specific cell types might be applicable to determine the embryonic/developmental toxicities of various toxicants and allow the comparison of the toxic stress depending on the stage of differentiation. Indeed, the search for stem cell–based assays has been recently attempted by others using mouse ES cell–derived cardiac, neuronal, and pancreatic cells as model systems to examine the toxicological effects (Rolletschek et al., 2004; Seiler et al., 2004).
In the present study, we provide additional evidence that hES cells can be useful for the study of developmental toxicity. In particular, we demonstrate for the first time that environmental toxicants, NP and OP, induce apoptosis in hES cells in a dose-dependent manner and that the Fas and FasL pathway is involved in this process. Our results also suggest that the response and sensitivity to the toxicants may be different depending on the status of differentiation. These findings offer new perspectives for understanding the fundamental mechanism of chemical-induced apoptosis in hES cells and might contribute to the development of a new predictive screening method for the hazard assessment of developmental toxicity using stem cell technology.
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NOTES |
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Disclaimer: The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
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ACKNOWLEDGMENTS |
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This research was supported by a grant (SC2050) from Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology to Dr J.H.K. and from Korean Research Foundation funded by Minister of Education and Human Resources Development (KRF-2003-C00051) to Dr Y.D.Y. All experimental procedures involving hES cells were approved by the Ministry of Health and Welfare and Korean Stem Cell Research Center (IRB number, 24).
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