ToxSci Advance Access originally published online on October 4, 2007
Toxicological Sciences 2008 101(1):81-90; doi:10.1093/toxsci/kfm256
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The Role of Mitochondrial and Oxidative Injury in BDE 47 Toxicity to Human Fetal Liver Hematopoietic Stem Cells
Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington 98105
1 To whom correspondence should be addressed at School of Public Health and Community Medicine, Department of Environmental and Occupational Health Sciences, 4225 Roosevelt Way NE, Suite 100, University of Washington, Seattle, WA 98105-6099. Fax: (206) 543 8458. E-mail: evang3{at}u.washington.edu.
Received July 27, 2007; accepted September 19, 2007
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ABSTRACT |
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The polybrominated diphenyl ethers (PBDEs) are a group of flame retardants whose residues have markedly increased in the environment and in human tissues during the last decade. Of the various congeners, BDE 47 (2,2',4,4'-tetrabromodiphenyl ether) is typically the predominant congener observed in fish and wildlife samples, as well as in human tissues. Several studies indicate in utero transfer of PBDEs during pregnancy with residues accumulating in fetal tissues, and thus the potential for BDE 47–mediated injury in utero is of concern. In this study, we examined the mechanisms of BDE 47–mediated injury to primary human fetal liver hematopoietic stem cells (HSCs), which comprise a large proportion of fetal hepatic cells and play a key role in hematopoiesis during fetal development. Incubation of fetal liver HSCs with BDE 47 led to a loss of mitochondrial membrane potential and the onset of apoptosis. These effects were observed in the low micromolar range of BDE 47 exposures. At higher concentrations, BDE 47 elicited a loss of viability, which was accompanied by the generation of reactive oxygen species and peroxidation of HSC lipids. Preincubation of fetal liver HSCs with N-acetylcysteine, a glutathione (GSH) precursor, caused an increase in cellular GSH concentrations, restored mitochondrial redox status, and ameliorated the toxicity of BDE 47. BDE 47–mediated cytotoxicity or oxidative injury was not evident at the lower concentrations (< 1µM). Collectively, these data support a role for oxidative stress in the cytotoxicity of BDE 47 and indicate that oxidative stress–associated biomarkers may be useful in assessing the sublethal effects of BDE 47 toxicity in other models. However, the fact that BDE 47 undergoes a concentration-dependent accumulation in other primary cells in media that can underestimate cellular concentrations (W. R. Mundy et al., 2004, Toxicol. Sci. 82, 164–169) suggests that the HSC cell injury observed in our study may be of less relevance to human in utero PBDE exposures.
Key Words: hematopoietic stem cells; polybrominated diphenyl ethers; oxidative stress; lipid peroxidation; apoptosis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The polybrominated diphenyl ethers (PBDEs) represent an important group of industrial chemicals that have been massively produced and extensively used in plastics, textiles, furniture, and electronic devices (McDonald, 2002
). Global production of PBDEs has reached approximately 67,000 metric tons (148 million pounds) per year (McDonald, 2002). PBDEs are used as additive flame retardants in plastics to which they are not chemically bound and can thus leach from polymers and accumulate in environmental media (McDonald, 2002; Sjodin et al., 2003). PBDEs share structural similarity to the persistent polychlorinated biphenyls (PCBs) and have high heat stability, high lipid solubility, and low vapor pressure, which contribute to their environmental persistence and bioaccumulation (Darnerud, 2003). On the other hand, the extent of toxicity by PBDE congeners can be dependent upon conformational disparity, such as differences in ether linkage, or in the case of PCBs, the position and degree of halogenation (Wang et al., 2006). Of note are recent studies demonstrating relatively high concentrations of PBDEs in the edible portions of fish tissues (Hites et al., 2004; Schecter et al., 2004), which may results in dietary exposures and PBDE body burdens in humans (McDonald, 2002; Sjodin et al., 2003).
In contrast to the PCBs, whose levels in environmental samples are decreasing (Noren and Meironyte, 2000), PBDE residues in environmental media and in human tissues appear to be increasing (Hale et al., 2003). For example, PBDE congeners in human breast milk from Swedish women have increased exponentially over the last two decades (Lind et al., 2003; Sjodin et al., 1999), and studies in North American populations have demonstrated the presence of PBDEs in human breast milk, adipose tissue, and blood (Betts, 2002; Schecter et al., 2003). Interestingly, the levels of PBDE congeners in breast milk of North American women in the aforementioned study population reflect a body burden that far exceed those in the Scandinavian studies (Lind et al., 2003; Schecter et al., 2003; Sjodin et al., 1999). Furthermore, PBDE concentrations in maternal blood predict the level of fetal exposures for some BDE congeners (Mazdai et al., 2003), suggesting maternal transfer to the developing fetus during pregnancy. This observation is supported by rodent studies demonstrating hepatic enzyme induction of cytochrome P4501A (CYP1A)–associated ethoxyresorufin-o-deethylase activity and pentoxyresorufin-o-deethylase activity in the livers of fetal rats whose mothers received PBDE exposures (Zhou et al., 2001). More recently, Schecter et al. (2007) demonstrated the presence of several PBDE congeners in human fetal liver tissue obtained through elective termination of pregnancy, substantiating transplacental exposure to PBDEs in humans. Collectively, these data underscore the importance of investigating the observed biological effects of maternally transferred PBDEs during pregnancy and indicate that the fetal liver is a potential target of such exposures.
Of the various PBDE congeners, 2,2',4,4'-tetrabromodiphenyl ether (IUPAC, BDE 47) appears to be a predominant PBDE congener present in human and wildlife tissues in North America (Bi et al., 2006; Mazdai et al., 2003; Schecter et al., 2007). This occurs despite its minor contribution to global PBDE production and usage (Darnerud, 2003; Hale et al., 2003). Pharmacokinetic studies indicate that BDE 47 is well absorbed following oral exposure, undergoes relatively little metabolism, and accumulates in adipose tissue, liver, and skin (Staskal et al., 2006a,b). BDE 47 is a predominant congener in human fetal liver (Schecter et al., 2007) and is consistent with the hypothesis that there may be critical windows of BDE 47 toxicity during development because of the poor capacity of the fetus to excrete BDE 47 (Staskal et al., 2006a). Thus, major public health concerns surround are associated with exposures BDE 47 exposures during pregnancy and their potential to cause adverse effects during development (Branchi et al., 2003; Eriksson et al., 2001; Gill et al., 2004). Although the mechanisms responsible for PBDE-induced injury are not well understood, recent research has focused on the ability of PBDEs to disrupt thyroid hormone status, leading to abnormalities in fetal growth and development in laboratory animals (Birnbaum and Staskal, 2004; Branchi et al., 2003; Meerts et al., 2000; Stoker et al., 2004; Zhou et al., 2002). However, PBDE congeners also have the potential to cause oxidative damage (Fernie et al., 2005). For example, in ovo and post-hatch exposures of captive American kestrels to PBDE congeners (predominantly BDE-47, -99, -100, -153) induce hepatic oxidative stress as reflected by elevated GSSG:GSH ratios and lipid peroxidation (Fernie et al., 2005). In the aforementioned study, it appeared that kestrels do not have adequate cellular defense mechanisms to protect against PBDE-induced oxidative stress, despite a carotenoid-rich diet used in the laboratory study (Fernie et al., 2005).
In the current study, we have characterized the effects of BDE 47 exposure in human cultured fetal liver stem cells and investigated the hypothesis that oxidative stress may be involved in BDE 47 mediated human cell injury. Our cell model, human fetal liver hematopoietic stem cells (HSCs) are the progenitor cells responsible for hematopoiesis during development and comprise a large proportion of liver cell populations in utero (Tavassoli, 1991). Because HSCs form the basis for hematopoiesis and the developing immune system, transplacental exposure to chemicals (including PBDEs) may result in adverse effects that manifest later in life (Alexander et al., 2001; Ma et al., 2002). In particular, HSC injury has been tied to the onset of hematopoietic disorders such as infant acute leukemia, possibly due to the essential roles of these cells in the normal development of hematopoiesis and immune function (Tavassoli, 1991). HSCs are sensitive to the toxicity of a number of compounds of relevance, including certain pesticides (e.g., chlorpyrifos, unpublished observations), carcinogens (e.g., etoposide, Moneypenny et al., 2006), and products of oxidative stress (e.g., 4-hydroxynonenal, Moneypenny and Gallagher, 2005).
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MATERIALS AND METHODS |
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Chemicals and biochemicals.
Iscove's modified Dulbecco's medium, penicillin, streptomycin, heat-inactivated fetal bovine serum, and AlamarBlue were purchased from Biosource (Invitrogen, Carlsbad, CA). Recombinant human interleukin 3 (IL-3), recombinant human granulocyte colony-stimulating factor (G-CSF), and recombinant human stem cell factor (SCF) were obtained from Research Diagnostics Inc. (Flanders, NJ). CD34 isolation columns were purchased from Miltenyi Biotec (Auburn, CA). Histopaque©-1977 was obtained from Sigma (St Louis, MO). Vented culture flasks and 96-well microtiter plates were purchased from Corning Inc. (Corning, NY). Dimethylsulfoxide (DMSO), N-acetylcysteine (NAC), and trypan blue were purchased from Sigma. BDE 47 (> 99% purity) was purchased from Chem Service, Inc. (West Chester, PA). BODIPY FL C11 (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid), Annexin V Alexa Fluor 350 conjugate, DCFH-DA (6-carboxy-2',7'-dichlorodihydrofluorescin diacetate), and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolo-carbocyanine iodide) were obtained from Molecular Probes/Invitrogen. All other chemicals were also obtained from Sigma or Fisher Scientific (Pittsburgh, PA).
Human liver tissues and isolation of CD34+ HSCs.
All use of human tissues was approved by the University of Washington Institutional Research Board and they were provided by the University of Washington Birth Defects Research Laboratory. Human fetal liver–derived CD34+ HSCs were isolated from second trimester fetal livers (typically 13–16 weeks of gestational age, but rarely from first trimester fetal livers of less than 12 weeks of age) as previously described (Shao et al., 2007). The freshly isolated liver tissue was dissociated under sterile conditions, and the resulting total fetal liver cell populations (including hepatocyte and nonhepatocyte fractions) were washed several times in 1x phosphate-buffered saline (PBS) buffer supplemented with 0.3% bovine serum albumin, 2.5 µg/ml amphotericin B-Fungizone, and 50 µg/ml gentamicin sulfate. The mononuclear layer was collected by centrifuging over 1.077 g/ml Histopaque©-1977 at 400 x g for 30 min at room temperature. The CD34+ cells were enriched by magnetic bead separation (Miltenyi Biotec). Our laboratory as well as others have observed that this cell preparation technique typically results in > 98% purity of CD34+ cells (de Wynter et al., 1998; Muench et al., 2002).
Cell culture and viability experiments.
HSCs were seeded at approximately 6250 cells/ml of Iscove's modified Dulbecco's medium containing 15% heat-inactivated fetal bovine serum, 2 ng/ml IL-3, 1 ng/ml G-CSF, 20 ng/ml SCF, 100 U/ml penicillin, and 100 µg/ml streptomycin (Moneypenny et al., 2006; Shao et al., 2007; Warren et al., 1995). The cells were maintained in culture for 7 days before treatment in a humid chamber at 37°C in 95% O2/5% CO2. This initial culture period allowed for a moderate increase in cell number while maintaining a stem cell phenotype (Moneypenny et al., 2006; Warren et al., 1995). Cell counts and viability were determined over the culture period using a hemocytometer and trypan blue exclusion, respectively, as described below.
In initial dose-response experiments, 1 x 106 CD34+ cells were removed on day 7 and placed into wells of a 6-well culture plate. BDE 47 was added at concentrations ranging from 0.2 to 50µM, and controls received an equivalent amount of vehicle (0.2% DMSO vol/vol). The cells were then cultured for 24 h, and the effect of BDE 47 on cell viability was examined by two methods, including trypan blue exclusion and AlamarBlue reduction (Ahmed et al., 1994). For the AlamarBlue assay, 10,000 cells in 200 µl culture medium from each treatment (or controls) were plated in 96-well plates in three replicates and incubated with 20 µl AlamarBlue for 2 h, a time point that was predetermined in preliminary studies to be optimal for assessing treatment-related effects on viability. The change in fluorescence associated with AlamarBlue reduction was recorded at 530ex/590em on a SpectraMax Gemini XS fluorometric plate reader (Molecular Devices, Sunnyvale, CA).
Effects of BDE 47 on mitochondrial injury and apoptosis.
To determine the effect of BDE 47 on mitochondrial membrane potential and apoptosis, HSCs were cultured as described above and transferred to 25-cm2 flasks prior to the addition of 0–50µM BDE 47 as described in the tables and figures. Twenty-four hours following treatment, 1 x 106 cells were removed from each treatment condition for flow cytometry analysis. The effect of BDE 47 on mitochondrial membrane potential was analyzed by flow cytometry using the fluorescent dye JC-1. This technique is used for the detection of mitochondrial depolarization, which results in a decreased red/green fluorescence intensity ratio and often corresponds to mitochondrial injury and the initiation of apoptosis (Chaoui et al., 2006; Lugli et al., 2005; Poot and Pierce, 1999). Specifically, JC-1 localizes to the mitochondrial inner membrane forming either aggregates (reflecting a high membrane potential as indicated by red fluorescence) or monomers (reflecting a low membrane potential and fluorescing green) depending on membrane potential (White and Reynolds, 1996). JC-1 was prepared in DMSO and added to the cell suspension at a final concentration of 1 µg/ml for 15 min at 37°C prior to rinsing with PBS and fixation for 10–30 min with 4% paraformaldehyde. The fluorescence intensities were acquired on a Coulter Elite flow cytometer (Beckman Coulter, Miami, FL) using 488 nm excitation. Green (JC-1 monomer) and red (JC-1 aggregate) emission characteristics were acquired with 525/40-nm band-pass (green) and 590-nm long-pass (red) filters, respectively.
To test the ability of BDE 47 to induce apoptosis, HSCs were treated with various concentrations of BDE 47 for 24 h and resuspended in Annexin V binding buffer (10mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 140mM NaCl, and 2.5mM CaCl2, pH 7.4) at a density of 1 x 106 cells/100 µl. The cells were then labeled with Annexin V Alexa Fluor 350 conjugate, which labels apoptotic cells by binding to phosphatidylserine translocated from the inner to the outer leaflet of the plasma membrane during apoptosis (van Engeland et al., 1998). Cells were then fixed in 4% paraformaldehyde for 10 min, washed once with 1x PBS, and analyzed by flow cytometry using 355 nm excitation. Blue fluorescence emission was acquired through a 450/35-nm band-pass filter.
Analysis of BDE 47–mediated cellular oxidative injury.
The potential for BDE 47 to stimulate reactive oxygen species (ROS) production in HSCs was evaluated using the oxidation of DCFH-DA to the highly fluorescent compound 2,7-dichlorofluorescein (DCF) in 96-well plate format (Krejsa and Schieven, 2000). DCFH-DA oxidation has been shown to be related to the amount of cytochrome c released from mitochondria into the cytosol of cells undergoing apoptosis, where it acts as a peroxidase (Lawrence et al., 2003). Briefly, 10,000 cells were incubated in 96-well plates in the presence of 10µM DCFH-DA under normal growth conditions for 30 min at 37°C. The cells were then exposed to BDE 47 as described above using eight replicates for each BDE 47 concentration. The rate of DCFH-DA oxidation to DCF was measured at 30 and 60 min postexposure using a SpectraMax Gemini XS fluorometric plate reader (Molecular Devices) at 485ex/530em (Grzelak et al., 2001). Hydrogen peroxide (H2O2) at 2mM level was used as positive control for ROS generation, as DCFH-DA oxidation is primarily facilitated by H2O2, which relies on peroxidase activity (Hirabayashi et al., 1985).
To determine the effect of BDE 47 on oxidative damage to lipids, HSCs were cultured as described above and transferred to 25-cm2 flasks and exposed to BDE 47. Twenty-four hours following dosing, the cells were incubated in the presence of 0.5 µg/ml BODIPY FL C11, a fluorescent dye that localizes to cell membranes (Pap et al., 2000). Cells were then rinsed with PBS and fixed with 4% paraformaldehyde for 30 min on ice. The intensity of BODIPY FL C11 green fluorescence was acquired on the flow cytometer using 488 nm excitation. As BODIPY FL C11 is oxidized, there is an associated reduction in the number of C11 excimers (yellow-red fluorescence) and a relative increase in the numbers of C11 monomers (green). In this assay, carbon tetrachloride (CCL4) was used as positive control for lipid peroxidation as CCL4 is a well-characterized xenobiotic for inducing lipid peroxidation in hepatotoxicity (Bhat and Madyastha, 2000; Lee et al., 2003).
A subset of BDE 47–exposed HSCs were analyzed for glutathione status (including reduced form [GSH], the oxidized form [glutathione disulfide, GSSG], and total glutathione [GSH + GSSG]) by a modification of the method of White et al. (2003). Following exposure, the cells were pelleted by centrifugation, resuspended in 20mM Tris, pH 7.4, 1mM ethylenediaminetetraacetic acid, 1.25M sucrose, 2mM L-serine, 20mM sodium borate, diluted 1:1 with 10% SSA (vol/vol), and sonicated. The cells were then centrifuged at 15,000 x g for 5 min, and a 25 µl aliqout of supernatant was added to 100 µl of 0.2M 4-ethylmorpholine/0.02M KOH and 10 µl of 10mM 2-carboxyethylphosphine hydrochloride in a fluorescence microtiter plate. Following a 15-min incubation at room temperature, 50 µl of 0.5N NaOH and 10 µl of 10mM 2,3-naphthalene dicarboxaldehyde in DMSO were added and samples were incubated for an additional 30 min in the dark. Fluorescence was then read at 472ex/528em on a Molecular Devices fluorometric plate reader. Cellular glutathione concentrations were evaluated against a standard curve prepared with reduced GSH. Protein concentrations were determined using the Bradford assay and a commercial kit (Sigma), using bovine serum albumin as a standard.
Effect of NAC on protecting against BDE 47–mediated oxidative damage.
To determine the effect of NAC on protecting against BDE 47–mediated cytotoxicity, two series of experiments were carried out. In the first experiment, HSCs prepared from pooled tissues were cultured in the presence of 5 or 8mM NAC to evaluate the potential for NAC to modulate cellular GSH concentrations. Following this initial experiment, HSCs were prepared from pooled tissues and cultured in the presence of 8mM NAC for 12 h. The cells were then centrifuged at 150 x g for 5 min and resuspended with fresh media containing either 0.2% DMSO or 50µM BDE 47 and cultured for an additional 24 h. At 24 h, the effects of NAC on cellular GSH concentrations, cell viability, and mitochondrial membrane potential were determined as described above.
Statistical analyses.
Experimental values for all oxidative stress and viability indices in the experiments represent the mean (SEM) of a minimum of triplicate replications performed in three experiments and using HSC preparations from different individual liver donors. The effects of NAC on BDE 47–mediated oxidative damage were investigated using triplicate incubations in two larger experiments that required pooled donors, with the results reported from a representative experiment. Treatment-related effects were assessed using one-way ANOVA followed by either Tukey's or Bonferroni's post hoc test. Differences were considered statistically significant at p 
0.05.
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RESULTS |
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Effect of BDE 47 on HSC Viability
As observed in Figure 1, exposure of HSCs to 50µM BDE 47 caused a significant loss of cell viability. The loss of cell viability on exposure to 50µM BDE 47 was evident by both AlamarBlue reduction and trypan blue exclusion assays although the magnitude of injury differed between the two methods tested. Specifically, an 8% loss in the number of live cells was observed using trypan blue exclusion (Fig. 1A), whereas a 25% loss in HSC viability was observed by the AlamarBlue assay (Fig. 1B).
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Effects on Mitochondrial Injury and Apoptosis
In flow cytometry experiments designed to test the effects of the BDE 47 on mitochondrial membrane depolarization, the treated cells fell into three distinct cell populations as identified by JC-1 staining 24 h following BDE 47 exposure. These included (1) a healthy cell population with high mitochondrial membrane potential which stained red, (2) a cell population with low mitochondrial membrane potential which stained green, and (3) a cell population with intermediate mitochondrial membrane potential which stained yellow (data not shown). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratios. As shown in Figure 2, a marginal decrease in JC-1 red/green ratios was observed at 0.8µM BDE 47 (a 20% decrease in red/green ratios relative to controls); however, a statistically significant decrease in mitochondrial membrane potential was not observed until cells were exposed to 12.5µM BDE 47, which caused a significant 30% decrease in mitochondrial membrane potential relative to control cells. Exposure to the higher dose of 50µM BDE 47 caused further loss in mitochondrial membrane potential, as reflected by a 39% decrease in red/green fluorescence ratios relative to controls.
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As shown in Figure 3, HSCs exposed to BDE 47 and analyzed for Annexin V staining by flow cytometry fell into two distinct populations, including a population of healthy cells that were Annexin V negative which localized to the left side of the cytogram. In contrast, Annexin V–positive cells, representing early and late apoptotic/necrotic cells, revealed a higher fluorescent intensity and were shifted to the right (Fig. 3, in the boxed area). In contrast to the other experimental analyses, we observed variability in Annexin V staining across experiments associated with baseline staining efficiencies with the fluorescent probe and possibly differences in the sensitivity of cells from three different donors to BDE 47 exposure. Accordingly, the results of three experiments are reported separately in Table 1. For donor 1, exposure to 50µM BDE 47 led to a 43% increase in the percentage of HSCs exhibiting positive Annexin V staining compared to controls (Table 1). In contrast, significant increases in apoptosis were not observed at BDE 47 doses below 50µM. In HSCs from donor 2, exposure to 12.5 and 50µM BDE 47 caused a significant increase in apoptosis (Table 1). HSCs analyzed from donor 3 displayed less overall Annexin V staining than those from the other two individuals in the absence of test chemical. However, HSCs from donor 3 were the most sensitive to treatment, with significant increases in apoptosis observed with exposure to as low as 3.2µM BDE 47 (Table 1).
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BDE 47 Stimulation of ROS Production
As shown in Table 2, exposure to BDE 47 stimulated an overall dose-dependent increase in ROS generation. At the 1-h post-exposure time point, a statistically significant stimulation of ROS production was observed for all BDE 47 exposures (Table 2). In particular, the level of ROS produced at 50µM BDE 47 was consistently higher than for the controls and for other BDE 47 treatments at both time points. Specifically, a 17% increase in ROS production was observed at 30 min, whereas a 21% increase in ROS production was observed at 1 h (Table 2). The positive control of 2mM H2O2 used in this assay led to very dramatic increase in ROS production (Table 2).
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Effect of BDE 47 on Lipid Peroxidation
BDE 47 treatment was associated with increasing BODIPY FL C11 fluorescence compared to the control cells receiving vehicle (Fig. 4). Since the assay was done in HSCs from different donors in different experiments, the baseline fluorescence intensities were subject to individual variation and the data are reported as percent increase relative to controls. A significant increase in green fluorescence was observed at 50µM BDE 47 (24% increase) compared to DMSO control, which is consistent with an increase in the oxidation of this fluorophore.
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Effect of NAC on BDE 47–Mediated Mitochondrial and Oxidative Injury
As shown in Figure 5A, HSCs incubated with 5mM NAC for 12 h exhibited 20% and 24% increases in reduced and total glutathione concentrations, respectively. In addition, this treatment reduced the percent of GSSG (as a percentage of total glutathione) from 2.6% to 0.9%. Incubation of cells with a higher dose of 8mM NAC did not further stimulate an increase in cellular GSH concentrations but further reduced the percentage of total glutathione accounted for by GSSG to 0.2%. Accordingly, the higher concentration of 8mM NAC was selected for a subsequent chemoprotection experiment. As observed in Figure 5B, pretreatment of HSCs with 8mM NAC protected against the loss of viability elicited by exposure to 50µM BDE 47. Because of the importance of JC-1 flow cytometry as an early indicator of BDE 47–mediated cell injury, this endpoint was used to determine if the protective effects of NAC could be associated with the restoration of mitochondrial membrane potential. As observed in Figure 5C, the 36% depolarization of the mitochondria on exposure to 50µM BDE 47 was reversed by preincubation with 8mM NAC. NAC pretreatment in the absence of test chemical did not affect HSC viability or mitochondrial membrane potential compared to non-pretreated controls (Figs. 5B,C).
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DISCUSSION |
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Collectively, laboratory studies using animal models, as well as epidemiological and human tissue studies, indicate that certain environmental chemicals and drugs can cross the placenta during pregnancy and interact with fetal cell targets and lead to disorders which arise later in development (Alexander et al., 2001
; Ma et al., 2002; Woodruff et al., 2004). In our laboratory, we have demonstrated that suspension culture of human fetal liver HSCs represents an appropriate in vitro model with relevance to the in utero environment (Moneypenny and Gallagher, 2005; Moneypenny et al., 2006; Shao et al., 2007). Of note is the fact that these human progenitor cells have relatively low constitutive CYP biotransformation capacity under our culture conditions compared to CYP levels in human fetal liver (Shao et al., 2007). This is consistent with in vitro studies of human stem cells from other cellular origins, including bone marrow (Bernauer et al., 2000; Kousalova et al., 2004). We also have observed in unpublished studies that these cells may have a relatively low capacity to undergo DNA repair following DNA damage. Accordingly, the progenitor nature of these cells may help to explain their sensitivity to environmental chemical injury. In the current study, our primary objective was to determine the relative susceptibility of these cells toward BDE 47 and investigate mitochondrial injury and oxidative stress as potential mechanisms of BDE 47–induced injury.
Interestingly, the AlamarBlue assay used in our study revealed a more dramatic decrease in cell viability at the 50µM BDE 47 exposure levels relative to the trypan blue assay. AlamarBlue is a redox-sensitive dye, in that the level of its reduction reflects cellular metabolic activity associated with mitochondria (Ahmed et al., 1994) and therefore serves as an indirect indicator of chemical-induced cytotoxicity (Fields and Lancaster, 1993). In addition to reflecting viability status, AlamarBlue reduction reflects the integrity of oxidation-reduction activity of the respiratory chain, a major intracellular source of ROS (Lee and Wei, 2007). Accordingly, a loss in AlamarBlue fluorescence in some cases may not reflect cell death. In contrast, the trypan blue dye exclusion assay more directly reflects cell death since it measures the loss of plasma membrane integrity.
The aforementioned observations suggest that BDE 47 may have elicited mitochondrial injury through programmed cell death. In this regard, various stimuli, particularly oxidative stress, can lead to loss of integrity in the mitochondrial inner membrane, resulting in mitochondrial permeability transition, cytochrome c release, and activation of downstream effector caspases (Delhalle et al., 2003). These two apoptotic pathways are closely interconnected as the mitochondrial pathway is often required to amplify relatively weak death receptor–induced apoptotic signals (Gross et al., 1999). By examining mitochondrial membrane depolarization and Annexin V labeling, we tested the hypothesis that the observed loss of cell viability was associated with apoptosis and at least partially dependent upon oxidative injury originating from the mitochondria. Mitochondrial membrane depolarization and the subsequent release of proapoptotic factors such as apoptosis-inducing factor and cytochrome c into the cytosol are purported to be required for the activation of apoptosis (Delhalle et al., 2003; Green and Reed, 1998). A reduction in mitochondrial redox status, an early event that precedes permeability of cell membranes to propidium iodide, has been observed in cell models of apoptosis (Kroemer et al., 1997; Lugli et al., 2005).
The fact that we observed a trend in the loss of mitochondrial membrane potential at BDE 47 concentrations below those causing cytotoxicity supports our hypothesis that BDE 47 targets the mitochondria and likely stimulates apoptosis via mitochondrial oxidative injury (Lugli et al., 2005). Furthermore, the fact that BDE 47 elicited an increase in Annexing V labeling is consistent with the early phases of apoptosis prior to the decomposition of cell membranes. Changes in phosphatidylserine asymmetry occur prior to morphological changes associated with apoptosis and before compromised membrane integrity (van Engeland et al., 1998). However, it must be noted that Annexin V also labels internal phosphatidylserine in membrane-compromised cells, and therefore this assay measures both early and late apoptotic/necrotic cells.
The fact that BDE 47 stimulated the production of ROS and that cellular lipid peroxidation was observed in high concentrations of BDE 47 suggests a role for oxidative damage associated with the observed mitochondrial injury. An unstable mitochondrial membrane and redox transition can occur as a result of diverse pathological states as well as from toxicant exposures and lead to a loss of mitochondrial integrity and cell survival (Loh et al., 2006; Zorov et al., 2006). Mitochondria are a major source of intracellular ROS and can stimulate further ROS production when injured through the loss of cytochrome c to the cytosol, which can in turn activate induction of apoptosis (Delhalle et al., 2003; Szeto, 2006; Zorov et al., 2006). Accordingly, our observations of increased ROS generation at lower micromolar concentrations of BDE 47 preceding cell death in human fetal liver HSCs are consistent with the pathogenesis of oxidative stress. The overall level of lipid peroxidation in HSCs exposed to 50µM BDE 47 is consistent with our hypothesis that oxidative stress strongly contributes to BDE 47–mediated cell injury in HSCs (Pap et al., 2000).
To test the hypothesis that oxidative stress was actually a causal agent in the observed cell death, we conducted a chemoprotection study with NAC. This compound is a thiol-containing antioxidant that functions as a scavenger of ROS, a regulator of cellular redox status, and as a precursor for GSH synthesis (Zafarullah et al., 2003). Treatment with NAC inhibits apoptosis induced by a variety of stimuli (Konarkowska et al., 2005; Wu et al., 2005; Zachwieja et al., 2005). In the current study, we observed that pretreatment with NAC facilitated an increase in GSH production, attenuated BDE 47–induced loss of viability and mitochondrial membrane potential, and afforded protection against cell injury from BDE 47. Interestingly, the fact that we were able to facilitate an increase in GSH biosynthesis in HSCs indicates that the biochemical pathways for GSH synthesis via glutathione biosynthetic enzyme pathways can be stimulated in these progenitor cells.
Collectively, the duration and volume of exposure and percentage of serum in the medium can all influence the accumulation of BDE 47 in cells in vitro (Mundy et al., 2004). Exposure of rat primary neocortical cells in serum-free media for 60 min results in a 100-fold magnification of the applied concentration (e.g., a 60-min exposure to 1µM BDE 47 resulted in 100µM concentration in the cells) (Mundy et al., 2004). However, in the presence of 10% serum, cell BDE 47 concentrations were reduced by 80% resulting in a 20-fold magnification of concentration and 3% of the applied BDE 47 associated with the cells, with approximately 96% of the applied dose remaining in the medium. The presence of serum in our treatment media, which is necessary to maintain viability of HSCs, likely resulted in partitioning of most of the compound into serum components (e.g., lipid, albumin) and likely reduced the efficiency of cellular uptake (Mundy et al., 2004). However, even in the presence of serum, it is likely that the cellular concentrations of BDE 47 were well above those applied to the media and that media concentration was not a good surrogate of in vitro cellular concentrations. It is also important to note that we did not confirm our cellular BDE 47 concentrations in the present study and it is not known if uptake kinetics of BDE 47 in cultured HSCs is similar to those reported by Mundy et al. using rat neocortical cells.
Total PBDE concentrations in maternal and fetal sera using paired mother and fetal blood samples in one study were roughly 14–600 ng/g lipid, with BDE 47 accounting for approximately 50–60% of total PBDEs in the serum (Mazdai et al., 2003). Given the 0.5% lipid content in human serum (Papke et al., 2004), it is reasonable to assume that BDE 47 concentrations in fetal circulation would be roughly 0.07–2.5 ng/g wet weight or < 5nM (Mazdai et al., 2003), which is likely well below the range of concentrations present in our HSCs based upon in vitro to in vivo extrapolations and the above-mentioned partitioning considerations. Accordingly, the results of our study suggest that BDE 47 may not be as toxic to human HSCs under physiological conditions associated with in utero exposures. However, the fact that a number of factors, including duration and volume of exposure, as well as concentration of serum proteins can influence the accumulation of BDE 47 concentrations in cells indicates that further study is needed to carefully define relevant dose-response relationships for in vivo extrapolations. This notion is underscored by the fact that chronic low-level maternal and fetal exposures during pregnancy are more relevant than a single acute exposure. Our study provides a basis for future studies examining whether the mechanisms of acute injury observed in the present study are relevant to the lower chronic doses observed in utero.
In summary, our data indicate that BDE 47, a predominant PBDE congener found in biological tissues, causes cell injury to cultured human fetal HSCs and is associated with oxidative stress and mitochondrial injury. This compound appears to alter the mitochondrial membrane potential, facilitate the production of ROS, and enhance the peroxidation of cellular lipids. Although the resulting cell death occurs at a level that may be well above those concentrations likely to be of relevance to fetal exposures in utero, we have not examined the effect of multiple or chronic exposures which may be more relevant to the in utero environment. Also of interest are other pathways of cell injury, such as effects on differentiation pathways for hematopoietic cells that may possibly occur at much lower concentrations than cell death. The fact that we observed the onset of mitochondrial injury in the high nanomolar/low micromolar range of single doses to BDE 47 exposure is also noteworthy and may have implications for selection of biochemical endpoints in assessing the sublethal effects of these compounds. In addition, there is probably no environmental exposure scenario in which exposure to only the BDE 47 congener occurs. In this regard, little is known regarding the effects of PBDE mixtures relative to single compounds. Ongoing investigations in our laboratory are directed toward a better understanding of the mechanisms and relevant risks associated with PBDE exposures observed in utero.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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National Oceanic and Atmospheric Administration Coastal Ocean Program (NA05NOS4781253); National Institutes of Health (NIH HD000836).
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ACKNOWLEDGMENTS |
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Our gratitude was paid to the University of Washington Birth Defects Research Laboratory for providing human fetal livers. The technical comments of Dr Susan Tilton were greatly appreciated.
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