ToxSci Advance Access originally published online on June 11, 2008
Toxicological Sciences 2008 105(1):119-133; doi:10.1093/toxsci/kfn115
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Published by Oxford University Press 2008.
Development of a High-Throughput Screening Assay for Chemical Effects on Proliferation and Viability of Immortalized Human Neural Progenitor Cells
,1,2
* The Curriculum in Toxicology, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
2 To whom correspondence should be addressed at USEPA, Neurotoxicology Division, B105-05, Research Triangle Park, NC 27711. Fax: (919) 541-4849. E-mail: shafer.tim{at}epa.gov.
Received March 14, 2008; accepted June 5, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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There is considerable public concern that the majority of commercial chemicals have not been evaluated for their potential to cause developmental neurotoxicity. Although several chemicals are assessed annually under the current developmental neurotoxicity guidelines, time, resource, and animal constraints prevent testing of large numbers of chemicals using this approach. Thus, incentive is mounting to develop in vitro methods to screen chemicals for their potential to harm the developing human nervous system. As an initial step toward this end, the present studies evaluated an automated, high-throughput method for screening chemical effects on proliferation and viability using ReNcell CX cells, a human neural progenitor cell (hNPC) line. ReNcell CX cells doubled in
36 h and expressed the neural progenitor markers nestin and SOX2. High-throughput assays for cell proliferation (5-bromo-2'-deoxyuridine incorporation) and viability (propidium iodide exclusion) were optimized and tested using known antiproliferative compounds. The utility of this in vitro screen was evaluated further using a set of compounds containing eight known to cause developmental neurotoxicity and eight presumably nontoxic compounds. Six out of eight developmental neurotoxicants significantly inhibited ReNcell CX cell proliferation and/or viability, whereas two out of eight nontoxic chemicals caused only minimal effects. These results demonstrate that chemical effects on cell proliferation and viability can be assessed via high-throughput methods using hNPCs. Further development of this approach as part of a strategy to screen compounds for potential effects on nervous system development is warranted. Key Words: neural progenitor cells; human; high-throughput screening.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Current toxicity testing efforts rely heavily on whole animal-based approaches for hazard identification and dose-response assessments. Although providing useful information, this system struggles to address several competing demands: to characterize chemical toxicity in a large-scale manner while minimizing the cost, time, and number of animals utilized. Little toxicity data is available for approximately 82,000 chemicals already in use in commerce and the approximately 700 new chemicals introduced into commerce annually (GAO, 2005
; NRC, 2007). Current toxicity testing approaches based on whole animals cannot screen all chemicals for hazard identification in a timely and cost-effective manner. Thus, development of in vitro screens to facilitate prioritization of chemicals for further testing was recently cited as a critical need for toxicity testing by the National Research Council's (NRC) Committee on Toxicity Testing and Assessment of Environmental Agents (NRC, 2007). Moreover, utilizing human cells to screen chemicals for neurotoxicity and developmental neurotoxicity, areas of significant public concern, has been proposed as potentially advantageous over other mammalian and nonmammalian models (Coecke et al., 2007; Lein et al., 2007; NRC, 2007). Development of screening approaches for developmental neurotoxicity testing presents several significant challenges: (1) developing appropriate human cell-based models, (2) designing screening approaches that are accurate and biologically appropriate in vitro recapitulations of the complex processes of nervous system development, and (3) developing high-throughput screening approaches that are both time and cost efficient.
Human-derived neural progenitor cells (hNPCs) can be utilized to screen chemicals for potential to cause (hazard identification) developmental neurotoxicity and prioritize them for further testing (hazard characterization). Although work with hNPCs has largely focused on their use in replacement therapies for disease states of the central nervous system (Reubinoff et al., 2001; Richardson et al., 2006; Roy et al., 2000), recent studies demonstrate the utility of hNPCs in assessing neurotoxicity in vitro (Fritsche et al., 2005; Kim et al., 2006; Li et al., 2005; Yoo et al., 2005; Zeng et al., 2006) and provide support for their use in hazard identification screens for potentially developmentally neurotoxic chemicals. However, effects of a wide range of chemicals on hNPC function have yet to be systematically examined as part of a high-throughput screening approach. The number of existing hNPC models used as screens has also been limited (Klemm and Schrattenholz, 2004; Richards et al., 2004, 2006), due in part to the difficulty in expanding human cells while maintaining a stable phenotype across multiple passages. Thus, implementing an immortalized, multipotent hNPC line provide an ideal in vitro model for screening for potential developmental neurotoxicity (hazard identification).
ReNcell CX cells are an immortalized NPC line derived from the cortex a 14-week human fetus. These cells maintain their ability to proliferate in the presence of growth factors, and in the absence of growth factors differentiate into neuronal, astrocytic, and oligodendrocytic cell populations (Donato et al., 2007). Previous work utilizing this line is limited, but they have been shown to expand in a linear fashion and express the intermediate filament protein nestin, a marker of NPCs (Donato et al., 2007). Therefore, these cells may represent an appropriate hNPC model, but have yet to be evaluated for toxicity screening.
Another challenge to developing in vitro screens to identify potential developmental neurotoxicants is that the complex neurobiology underlying central nervous system development is not completely understood and cannot be completely modeled by an in vitro system. However, central nervous system development requires several global processes that can be assessed in vitro including, but not limited to, NPC proliferation, migration, and differentiation. Therefore, a suite of assays that can screen for chemical effects on these and other endpoints may provide an approach to screen and prioritize chemicals for additional testing (Coecke et al., 2007; Costa et al., 2007; Lein et al., 2005, 2007). Moreover, the demand to assess large numbers of chemicals for hazard identification in a cost- and time-efficient manner highlights the need for high-throughput assays. High-content approaches have recently progressed to exploit automated microscopy, particularly to evaluate cell proliferation during specific phases of the cell cycle (Barabasz et al., 2006; Gasparri et al., 2004; Grove and Ghosh, 2006). Thus, high-throughput/high-content screening approaches can be applied to evaluate chemical effects on cell proliferation.
Because of the importance of neuroprogenitor proliferation in nervous system development, and because compounds that inhibit proliferation can cause developmental neurotoxicity (Adlard et al., 1975; Kuwagata at al., 1998), an assay to assess chemical effects on proliferation would be an important component of a suite of assays to screen chemicals for hazard identification. Ideally, this would be a high-throughput, in vitro assay to minimize cost, time, and the use of animals, and would utilize neuroprogenitor cells (because of their ability to proliferate) derived from human tissue. Therefore, the present experiments were conducted to achieve three specific aims. First, to confirm the neural progenitor nature of ReNcell CX cells, the expression of two commonly measured markers of NPCs, the intermediate filament protein nestin (Lendahl et al., 1990) and transcription factor SOX2 (Graham et al., 2003) were examined. Next, assays to measure cell proliferation and viability utilizing a high-throughput/high-content approach were developed. To demonstrate the ability of this approach to detect inhibition of ReNcell CX cell proliferation, effects of growth factor withdrawal as well as exposure to known antiproliferative compounds was examined. Finally, to demonstrate that this approach could be utilized to examine effects of a series of chemicals over a wide concentration range, effects on proliferation and viability of ReNcell CX cells were examined following exposure to a set of 16 chemicals. This set included compounds known to cause developmental neurotoxicity in mammals and compounds generally perceived to be non-neurotoxic. The results demonstrate the feasibility of high-throughput/high-content approaches to identify chemicals that inhibit proliferation of hNPCs, and support further evaluation of this approach as part of a suite of assays to screen chemicals to identify potential neurotoxicants.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Cell culture.
ReNcell CX cells were obtained commercially from Millipore (Temecula, CA). This cell line was derived from a 14-week gestation human fetal cortex obtained from Advanced Bioscience Resources (Alameda, CA); details outlining its isolation have been described previously (Donato et al., 2007
). For all experiments, cells frozen at passage 3 were thawed and expanded on laminin-coated T75 cm2 tissue culture flasks (Corning, Inc., Corning, NY) in ReNcell NSC Maintenance Medium (Millipore) supplemented with epidermal growth factor (EGF) (20 ng/ml; Millipore) and basic fibroblast growth factor (FGF-2) (20 ng/ml; Millipore). Three to four days after plating (e.g., prior to reaching 80% confluency), cells were passaged by detaching with accutase (Millipore), centrifuging at 300 x g for 5 min and resuspending the cell pellet in fresh maintenance media containing EGF and FGF-2. For all experiments, cells were replated in laminin-coated costar 96-well plates (Corning, Inc., Corning, NY) at a density of 10,000 cells per well. Thus, for all experiments, cells from passage 4 were utilized; this prevents the possibility that changes in the characteristics of the cells over multiple passages might influence the results.
Immunocytochemical characterization of ReNcell CX cells.
Immunocytochemical experiments were conducted to determine whether undifferentiated ReNcell CX cells express the NPC markers nestin and SOX2. Cells were fixed with a 4% paraformaldehyde solution and permeabilized in blocking solution (5% normal goat serum, 0.3% Triton X-100 in phosphate-buffered saline). Nestin and SOX2 expression were determined by incubating cells overnight at 4°C in primary antibodies against nestin (1:1000, mouse anti-nestin, Chemicon, Temecula, CA) or SOX2 (1:200, rabbit anti-SOX2, Chemicon), and labeling with Alexa Fluor 488 (nestin) or 594 (SOX2). Cell nuclei were then labeled by counterstaining with Hoechst 33258 and visualized using a Nikon TE200 inverted fluorescence microscope with a 20x objective. Images were captured using an RT Slider camera (Model 2.3.1., Diagnostic Instruments, Inc., Sterling Heights, MI) and SPOT Advantage software (Version 4.0.9, Diagnostic Instruments, Inc.).
Assessment of cell proliferation.
ReNcell CX cell proliferation was determined by quantifying DNA replication in ReNcell CX cells using the Cellomics BrdU Cell Proliferation Kit for high-content screening (ThermoFisher Scientific, Pittsburgh, PA). 5-Bromo-2'-deoxyuridine (BrdU), a thymidine analog, enables the detection of actively proliferating cells that have progressed through S phase of the cell cycle. BrdU incorporation in ReNcell CX cells was quantified using a high-content analysis system (Cellomics Arrayscan VTI) with a cell-based assay in which cells are automatically imaged and analyzed using quantitative fluorescence microscopy. Cells were incubated with BrdU at a final concentration of 53µM for the final 4 h of the chemical exposure period (4, 24, or 48 h), and were then fixed with a 4% paraformaldehyde solution. After cell membrane permeabilization, cell nuclei were labeled blue using DAPI dye. BrdU incorporated into each cell was labeled red using a mouse anti-BrdU primary antibody and DyLight 549 conjugated goat anti-mouse IgG secondary antibody. Cells were then imaged and analyzed using the ArrayScan VTI high-content imaging system to provide the mean average intensity of fluorescently labeled BrdU within cell nuclei. Known antiproliferative compounds (Table 1) were chosen to optimize the proliferation assay and demonstrate the feasibility of using this method in RenCell CX cells in a high-throughput/high-content format.
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Assessment of cell viability.
For all experiments, ReNcell CX cell viability was determined by staining cells with propidium iodide (Invitrogen, Carlsbad, CA) and Hoechst 33258. Propidium iodide is a red fluorescent nuclear and chromosomal counterstain that is not permeant to live cells and therefore only stains cells with compromised cell membranes. Hoechst 33258 is a blue fluorescent stain that labels all cell nuclei. After chemical exposure, cell nuclei were labeled blue with 12 µg/ml Hoechst 33258 dye and dead cells were labeled red with 2.5µM propidium iodide (final concentration). Hoechst 33258 and propidium iodide were added directly to the media and incubated at 37°C for 15 min. Cells were then imaged using the Cellomics Arrayscan VTI high-content imaging system. Cell viability was calculated as the proportion of all cells not stained with propidium iodide.
Image acquisition and analysis.
For both proliferation and cell viability assays, the Cellomics ArrayScanTI high-content imaging system (ThermoFisher Scientific) was used to automatically focus, expose and acquire images using a 10x objective lens. Fluorescence images were generated using a multiple bandpass emission filter and matched excitation filters (XF93). Images were collected on two channels, one for nuclear labeling and one for BrdU incorporation (proliferation assay) or propidium iodide staining (viability assay). Valid objects were determined in the nuclear channel (channel one) and were subsequently analyzed in the second channel (channel two). Criteria for nuclear parameters were based on object intensity, area, and dimensions using accompanying algorithms. Objects not meeting criteria were excluded from analysis. The algorithm denotes these with a mask of a different color (orange), and these typically include cells that have clumped together or are not completely located within the field of view. Images from channel two were captured in gray scale and analyzed for mean average intensity (BrdU incorporation) and percent average intensity responders (propidium iodide) using the Target Activation bioapplication (ThermoFisher Scientific). A sufficient number of fields were imaged and analyzed to collect data from at least 200 cells per well. For BrdU incorporation, the mean average intensity within each cell nucleus in a given field was determined. To assess cell viability, the percentage of cells within a given field stained with propidium iodide was determined and subtracted from 100 percent to yield the percentage of viable cells. Algorithms used to evaluate both cell proliferation and viability were verified for accuracy on both channels. The masks generated in the nuclear channel (channel 1) were inspected manually to ensure that they enclosed only area stained by DAPI dye or Hoechst 33258. The same procedure was performed for channel 2 (BrdU or propidium iodide). The data produced by the algorithm was also verified manually by counting the number of positively stained cells for both channels as a percentage of the total population within a given field. Data are presented as mean average intensity (mean ± SD) for Figures 3A and 4A. For clarity, data for all other experiments are presented as percent control (mean ± SE) for both measures.
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Test chemicals examined for effects on proliferation and viability.
Dimethyl sulfoxide (DMSO), the vehicle for the majority of tested chemicals, was purchased from Sigma-Aldrich (St Louis, MO). A set of 16 commercially available, toxicologically diverse, test chemicals was chosen to develop a protocol for screening chemicals over a wide concentration range using the high-throughput/high-content screening system (Table 2). Eight chemicals were selected based on the availability of data demonstrating adverse effects on the developing nervous system. For these eight developmental neurotoxicants, a literature review confirmed evidence of neurotoxicity after developmental exposure in mammals, including studies in experimental animals and/or in humans. A second set of eight chemicals was selected based on the presumed absence of data indicating effects on the developing nervous system and/or approval for their use during pregnancy. Using PubMed, each chemical was searched along with the following terms: neurotoxicity, developmental neurotoxicity, proliferation, and cytotoxicity to find any peer-reviewed publications relevant to developmental neurotoxicity or the endpoints of proliferation and viability in neuronal preparations. For one compound, diphenhydramine, a report of developmental neurotoxicity in rats was found (Chiavegatto et al., 1997
); otherwise no relevant peer-reviewed publications for the second set of eight compounds were located in PubMed.
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Chemical preparation and exposure.
Chemicals were dissolved in either 100% DMSO (Sigma-Aldrich) or double-distilled H2O (ddH2O) based on the solubility of each chemical, and stock solutions of 1µM–100mM in semi-logarithmic increments were prepared for each chemical. For each experiment, 96-well stock plates were prepared by making 1:100 dilutions in media of each stock concentration of every chemical to be used in that experiment. RenCell CX cells were plated into 96-well plates in 90 µl of media. Sixteen hours after plating (to ensure cell adherence to the substrate), 10 µl of media (exposure media) from the 96-well stock plate (1:10 dilution) was added to the cells, with gentle pipeting to ensure mixing, to yield a final chemical concentration range of 1nM–100µM, with a final DMSO or ddH2O concentration of 0.1%. Glyphosate, lead and trans-retinoic acid were not soluble at 100µM therefore the highest concentration of these compounds tested was 30µM. Exposure media contained the growth factors EGF and FGF-2 in the same concentration as that present in wells housing cells. The arrangement of assay positive and negative controls and chemicals to be tested on each plate is discussed in the Results section. Cells were exposed to chemicals for 4, 24, or 48 h prior to assessment of proliferation or viability. Each well was considered an observation ("n") for statistical purposes, and data were collected from six wells on separate plates for each chemical at each concentration. To obtain the six plates, each experiment was repeated three times on separate days (hence cells were obtained from different replicate vials of the same passage) with two plates on each day.
Statistical analysis.
Chemical effects on cell proliferation and viability, as well as the effect of incubation time on growth factor removal-induced decreases in cell proliferation and viability, were determined using a one-way analysis of variance (ANOVA) followed by post hoc testing using Dunnett's post hoc test. Data obtained at each chemical concentration were compared with vehicle controls. For all comparisons, results were considered significant if p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Characterization of Undifferentiated ReNcell CX Cells to Confirm Neuroprogenitor Status
Undifferentiated ReNcell CX cells proliferated in a monolayer and displayed a rounded, immature neural morphology (Fig. 1A). Staining with Hoechst 33258 revealed that ReNcell CX cells proliferated in a linear fashion in peak log-phase growth with a doubling time of
36 h (Fig. 1A, inset). To confirm the progenitor nature of ReNcell CX cells, expression of the NPC markers nestin and SOX2 were determined using immunocytochemistry. ReNcell CX cells abundantly expressed both markers, with 100% of cells expressing both nestin and SOX2 (Figs. 1B–D).
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Development of Assays for Proliferation and Viability
Image acquisition.
Representative images presented in Figures 2A–C demonstrate how cell proliferation was evaluated using the Cellomics ArrayScan VTI. All cells in the field labeled with the nuclear dye DAPI are imaged in channel one (Fig. 2A). Algorithms are then used to determine valid objects for analysis and these were marked by blue masks outlining nuclei (Fig. 2B). Cells proliferating through the S phase of mitosis (BrdU-positive cells) were detected on channel 2 by the ArrayScan and accompanying software was utilized to overlay the images from channel 1 and 2 (Fig. 2C). The arrows in all three panels indicate cells that were positive for BrdU, whereas the circles designate cells that did not incorporate BrdU. BrdU incorporation was quantified using Cellomics bioapplication software to determine the average intensity obtained within each cell's nucleus (blue mask).
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For all experiments, cell viability was also determined using the ArrayScanTI in separate, identically treated 96-well plates. Detection of all cells was achieved by labeling cell nuclei with Hoechst 33258 (Fig. 2D). Algorithms generated blue nuclear masks and green masks larger in area than the nucleus (Fig. 2E) to enable complete quantification of propidium iodide staining within dead cells (Fig. 2F, denoted by arrows). Propidium iodide staining was quantified using Cellomics bioapplication software to count cells with an average intensity above a minimum threshold average intensity determined for healthy viable cells.
Growth factor and vehicle effects on ReNcell CX cell proliferation and viability.
The withdrawal of the growth factors EGF and FGF-2 from the media inhibits proliferation and induces differentiation in ReNcell CX cells (Donato et al., 2007). As a positive control for inhibition of cell proliferation, BrdU incorporation was assessed in cells maintained in media without growth factors. Growth factor withdrawal significantly inhibited BrdU incorporation by 43% after 24 h and 52% after 48 h (Fig. 3A). After 48 h, cell viability was also significantly decreased, albeit only by 7% (Fig. 3B). These data indicate that growth factor withdrawal selectively decreases proliferation at 24 h.
The vehicles for chemicals screened in the present experiments were either DMSO or ddH2O. To evaluate potential effects of DMSO on ReNcell CX cell viability, effects of DMSO (0–3%, vol/vol, final concentration) on propidium iodide staining were determined after a 48-h incubation. DMSO concentrations of >1% significantly increased propidium iodide staining (Fig. 4A). There was no change in propidium iodide staining in cells treated with DMSO concentrations between 0.1 and 0.5%. A concentration of 0.1% DMSO had no effect on BrdU incorporation after 4, 24, or 48 h (Fig. 4B) and therefore was used as a vehicle for all subsequent experiments. Preliminary experiments also revealed no difference between the degree of BrdU incorporation in control cells cultured in edge wells (mean average intensity 255.05 ± 14.12) compared with those cultured in inner wells (mean average intensity 238.12 ± 7.14). A final concentration of 0.1% ddH2O used as a vehicle for several compounds had no effect on BrdU incorporation or cell viability (data not shown).
Assessment of Known Proliferation Inhibitors on ReNcell CX Cell Proliferation and Viability
To demonstrate the ability of the ArrayScan high-content screening system to detect chemical effects on proliferation in ReNcell CX cells, the following antiproliferative compounds were examined for effects on BrdU incorporation: aphidicolin, hydroxyurea, cytosine arabinoside, 5-fluorouracil, and ochratoxin A (Table 1). Viability (propidium iodide staining) was also evaluated using the ArrayScan high-content screening system to determine the specificity of the antiproliferative action of each compound. Cells were exposed to chemicals for 4, 24, or 48 h to determine the effect of incubation time on proliferation. The DNA polymerase inhibitor aphidicolin (Urbani et al., 1995; Verri et al., 1994) inhibited BrdU incorporation into ReNcell CX cells at all incubation times, and the lowest effective concentration of 100nM for inhibition of proliferation was seen after 4 h, without any effect on cell viability at this time point (Figs. 5A–C). After 24 or 48 h, decreases in cell viability were observed at 100 and 1–100µM, respectively. These concentrations, however, were higher than those inhibiting cell proliferation for both time points. Hydroxyurea, which blocks DNA replication by inhibiting ribonucleotide reductase (Larsen et al., 1982), also inhibited BrdU incorporation at all three times, but cell viability was only decreased at 100µM after 24 or 48 h (Figs. 5D–F). Cytosine arabinoside, an inhibitor of cell proliferation by multiple mechanisms (Aguayo et al., 1975; Kizaki et al., 1992), inhibited BrdU incorporation at all three incubation times, but most efficiently after 48 h, with significant inhibition of proliferation seen as low as 3nM (Figs. 5G–I); cell viability decreased only after 24 or 48 h. The DNA synthesis inhibitor 5-fluorouracil (Imada et al., 1997) was without effect on RenCX cells after 4 h, but reduced BrdU incorporation and decreased ReNcell CX cell viability after 24 or 48 h (Figs. 5J–L) of exposure. Finally, ochratoxin A, which damages DNA through pro-oxidant mechanisms (Sava et al., 2007), inhibited BrdU incorporation at all incubation times and decreased cell viability after 24 or 48 h (Figs. 5M–O). Thus, after 4 h, four of the five known antiproliferatives inhibited BrdU incorporation while having no effect on cell viability. After 48 hrs, all five compounds decreased both BrdU incorporation and cell viability. None of the compounds decreased viability by greater than 17% at the highest concentration tested after 24 h. This resulted in a clear difference in the magnitude of inhibition seen for proliferation and viability for four of the five known antiproliferatives at this time point. Because there was also a significant decrease in BrdU incorporation without an affect on cell viability after growth factor removal (Fig. 3) after 24 h, this exposure time was chosen for all subsequent experiments.
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Screening for Chemical Effects on ReNcell CX Cell Proliferation and Viability
The plate map showing concentration ranges of chemicals assessed in subsequent experiments, as well as plate wells allotted for control conditions, is shown in Figure 6. Wells treated with the known DNA polymerase inhibitor aphidicolin (100µM) and the removal of growth factors FGF-2 and EGF served as internal plate positive controls, and those treated with 0.1% DMSO served as vehicle controls. All control conditions were situated in column 6. Semilogarithmic chemical concentrations of 1nM–100µM for eight different chemicals per plate (one chemical per row) were situated in columns 1–5 and 7–12.
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As an initial assessment of the utility of the ArrayScanTI high-content system as a screen for chemical effects on cell proliferation, effects on ReNcell CX cell proliferation and viability were determined for a set of 16 compounds (Table 2). Eight of these compounds were known developmental neurotoxicants, selected on the basis of available data in mammals and/or humans. The other eight compounds were selected on the basis of presumed lack of neurotoxicity and/or approval for use during pregnancy. Only for diphenhydramine did a review of the literature indicate any evidence of neurotoxicity. Of the eight chemicals for which in vivo evidence of developmental neurotoxicity exists, methyl mercury, trans-retinoic acid, cadmium, dexamethasone, lead, and D-amphetamine sulfate decreased BrdU incorporation into ReNcell CX cells after 24 h (Figs. 7A–F). Only three of these six chemicals (methyl mercury, trans-retinoic acid, cadmium) decreased cell viability; D-amphetamine, dexamethasone (only significant at 100µM) and lead inhibited cell proliferation without affecting cell viability. Of the chemicals that decreased cell viability, cadmium was the only compound to inhibit cell proliferation at concentrations (3–10µM) where cell viability was unaffected (Fig. 7D). Only two of the eight developmental neurotoxicants, 5,5-diphenylhydantoin and valproic acid, did not affect BrdU incorporation or viability (Figs. 7G and 7H).
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Of the eight non-neurotoxic chemicals screened, only omeprazole, at concentrations of 30 and 100µM, decreased BrdU incorporation in ReNcell CX cells (Fig. 8H). Diphenhydramine and omeprazole decreased cell viability after 24 h at 100µM (Figs. 8G and 8H). The remaining six non-neurotoxic compounds (amoxicillin, acetaminophen, saccharin, D-sorbitol, dimethyl phthalate, and glyphosate) did not affect proliferation or viability (Figs. 8A–8F). Similar effects were observed with this set of 16 compounds when examined using 4- or 48-h incubations (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The findings presented herein describe the development of a high-throughput chemical screen for cell proliferation in an immortalized human NPC line. ReNcell CX cells were characterized in the undifferentiated state and expressed the appropriate markers nestin and SOX-2. The feasibility of using this cell line in high-throughput/high-content screens for proliferation and cell viability was demonstrated using a set of compounds known to inhibit cell proliferation and by demonstrating that effects of a set of 16 chemicals on these endpoints could be assessed over a wide concentration range. Together, the results demonstrate that such an approach may be a useful component of a suite of assays to identify and prioritize chemicals for additional developmental neurotoxicity testing.
RenCellCX Cells Express Neuroprogenitor Characteristics
The present study demonstrated that ReNcell CX cells proliferated linearly, had an immature neuronal morphology, and coexpressed nestin and SOX2. These results are consistent with previous studies characterizing ReNcell CX cells and demonstrating genotypic stability of this line (Donato et al., 2007). Moreover, coexpression of nestin with SOX2, a transcription factor expressed by immature NPCs that inhibits differentiation (Graham et al., 2003) has not previously been reported in ReNcell CX cells. The withdrawal of the growth factors EGF and FGF-2 in the present studies also decreased BrdU incorporation as the incubation time increased, consistent with previous work showing that these progenitors begin to differentiate in the absence of growth factors (Donato et al., 2007). Together, these characteristics demonstrate that undifferentiated ReNcell CX cells represent an immortalized line of a true progenitor state and indicate that they may be a useful hNPC model for screening purposes.
ReNcell CX Proliferation Can be Assessed Using a High-Throughput/High-Content Approach
The large number of chemicals that need to be prioritized for developmental neurotoxicity testing highlights the need for high-throughput screens that are cost and time efficient. The present work demonstrated that BrdU incorporation into DNA of RenCell CX cells can be assessed using an automated, high-throughput approach. All five compounds known to inhibit cell proliferation in neural cells (Table 1) decreased BrdU incorporation into ReNcell CX cells with minimal decreases seen in cell viability. It is noteworthy that for four of these five compounds, effects on the developing nervous system are reported following developmental exposure in vivo. This underscores the importance of proliferation of neuroprogenitor cells in the developing nervous system.
The present findings also demonstrate the utility of the Cellomics ArrayScan to measure proliferation via BrdU incorporation and are consistent with previous work in which stimulation of cell proliferation using growth factors was detected in normal human dermal fibroblasts by this method (Gasparri et al., 2004). This previous work (Gasparri et al., 2004) also confirmed proliferation using enzyme-linked immunosorbent assay (ELISA) and flow cytometry, demonstrating the validity of the measurements using the Arrayscan. Other high-throughput methods have been utilized to assess cell proliferation, including ATP bioluminescence (Crouch et al., 1993), total protein quantification by sulforhodamine B (Skehan et al., 1990; Voigt, 2005), [3H]thymidine incorporation (Cleaver, 1965), or BrdU incorporation measured by ELISA (Perros and Weightman, 1991). However, these approaches have yet to be implemented to screen for chemical effects on proliferation in conjunction with assessment of cell viability. Several other studies have demonstrated chemical effects on proliferation of hNPCs (Fritsche et al., 2005; Kim et al., 2006; Li et al., 2005; Yoo et al., 2005; Zeng et al., 2006), but have not utilized a high-throughput/high-content approach.
The results of proliferation and cytotoxicity assays from three different time points indicated that assessment of effects following 24 h of treatment provided consistent results for most compounds. Based on these results, a 24-h exposure period was selected for subsequent experiments. Although assessment of multiple time points provides additional data, the time and materials (thus the cost) needed increases considerably when evaluating multiple time points. Therefore, it is more advantageous to evaluate chemical effects at one time point from a screening standpoint. This is especially the case when using assays that require fixing the cells, such as the proliferation assay.
Effects of a Set of Test Chemicals on Proliferation and Viability in ReNcell CX Cells
The present results also demonstrate that this approach can be used to evaluate effects of a toxicologically diverse series of chemicals on proliferation and viability. It is important to note that the purpose of examining this set of chemicals was to demonstrate the feasibility of these assays to examine multiple chemicals over a wide concentration range, rather than to determine the prediction rates for the assay. This approach was selected because for environmental chemicals, the concentrations that will be active are often unknown, and determination of concentration-response relationships provides greater biological information than assessment of single concentrations of a compound, an approach often used in pharmaceutical contexts (Xia et al., 2008). An upper limit of 100µM was selected because many environmental chemicals are not soluble above this concentration.
Of the 16 chemicals examined, seven had previously been reported to inhibit proliferation in neural cells (dexamethasone, 5,5’ diphenylhydantoin, lead, methyl mercury, trans-retinoic acid, valproic acid and acetaminophen). In the present study, 8 of the 16 chemicals examined decreased proliferation and/or viability of the ReNcell CX cells. Three of these eight chemicals were metals: methyl mercury, cadmium, and lead. Data for methyl mercury and lead are consistent with previous work in neural models (Burke et al., 2006; Costa et al. 2007; Huang and Schneider, 2004; Ponce et al., 1994). For cadmium, effects on proliferation had not been previously reported in neural models in vitro. However, this heavy metal has been widely reported to inhibit proliferation in non-neural models (Goldberg et al., 1983; Piersma et al., 1993). Three other compounds also inhibited proliferation; D-amphetamine, dexamethasone, and trans-retinoic acid. No reports of D-amphetamine on neural cell proliferation in vitro were found in the peer-reviewed literature. However, this compound has been reported to inhibit thymidine incorporation into DNA following developmental exposure to high doses (Béndek and Hahn, 1981). The present results with dexamethasone are consistent with its inhibition of proliferation in rat neuroprogenitor cells in vitro (Sundberg et al., 2006), and the decrease in proliferation observed in the present experiments following trans-retinoic acid treatment is consistent with the ability of this compound to induce differentiation in a wide variety of neural model systems and embryonic stem cells (Martín-Ibáñez et al., 2007; Rajasingh and Bright, 2006).
Two compounds for which effects on proliferation in neural cells were reported in the literature, 5,5-diphenylhydantoin and valproic acid, were without effect on ReNcell CX cell proliferation and viability. Valproic acid inhibited cell proliferation in primary cortical neurons without affecting cell viability (Regan et al., 1990) and inhibited proliferation of human NT2 cells (Skladchikova et al., 1998), C6 glioma cells (Courage-Maguire et al., 1997), and human neuroblastoma cells (Cinatl et al., 1996). The most potent IC50 value reported for inhibition in any of these studies was 800µM, eightfold higher than the highest concentration tested in the present study. By contrast, 500µM valproic acid stimulated proliferation of rat cortical NPCs (Laeng et al., 2004). Thus, effects of valproic acid on proliferation generally require higher concentrations than tested in the present studies and have been reported to cause either inhibition or stimulation of proliferation. 5,5’-Diphenylhydantoin inhibits proliferation of mouse primary astrocytes at concentrations > 50µM (Meyer et al., 2001), but has been reported to lack antiproliferative activity in a variety of primary and clonal neural culture models (Regan et al., 1990). Thus, the lack of effect of these two compounds under the conditions employed in the present assays are not necessarily inconsistent with previous results and may be due to the concentration range tested.
Of the other compounds screened in proliferation and viability assays, only omeprazole and diphenhydramine produced significant effects. Review of the peer-reviewed literature did not indicate any previous reports of effects of these compounds on proliferation or viability of neural cells in vitro. However, omeprazole has been reported to inhibit proliferation of human B-cells at concentrations >70µM (De Milito et al., 2007) and induce apoptosis in jurkat cells (Scaringi et al., 2004). Moreover, the antihistamine diphenhydramine inhibits proliferation of human immortalized lymphocytes (Malaviya and Uckun, 2000) and induces apoptosis in two human T-cell acute lymphoblastic leukemia cell lines (Jangi et al., 2004). The present results are consistent with these effects; omeprazole decreased cell proliferation and viability at concentrations > 30µM, whereas diphenhydramine decreased cell viability by only 10%, and only at the highest concentration tested, 100µM. For acetaminophen, there was a single report of effects of this compound on proliferation of rat and human glioma cells at less than millimolar concentrations (Bernardi et al., 2006). However, this was inconsistent with reports in other types of neural cells wherein 5–10mM acetaminophen was required to inhibit proliferation or reduce viability (Casper et al., 2000; Holownia et al., 1998; Mannerström et al., 2006). The lack of effect of acetaminophen on proliferation and viability in the present studies is consistent with these reports.
Including the known antiproliferative compounds in Table 1, there were twelve compounds that had been previously reported to inhibit neuronal proliferation in vitro. Nine of these compounds inhibited proliferation in the present study. For the three compounds that did not (diphenylhydantoin, valproic acid, and acetaminophen), the concentrations reported in the literature to inhibit proliferation were higher than those tested in the present study and for valproic acid, reports in the literature regarding proliferation of neural cells are equivocal. Thus, the assay is relatively robust at detecting compounds that inhibit proliferation; particularly those that do so in the low micromolar concentration range. The present studies also identified cadmium, D-amphetamine and omeprazole as inhibitors of proliferation in hNPCs.
The inclusion of viability measures and concentration-response assessments in the present studies provide additional biological information to screen and prioritize chemicals. Of the 16 compounds tested, five were specific inhibitors of proliferation (D-amphetamine, lead, dexamethasone, cadmium, and omeprazole), causing a significant decrease in proliferation at concentrations lower than those causing a significant decrease in viability. Additionally, methyl mercury and trans-retinoic acid were less specific inhibitors of proliferation, causing significant decreases in proliferation in conjunction with significant decreases in viability. However, both of these compounds caused nearly complete inhibition of proliferation at the highest concentrations tested. It is not clear from the present data whether the antiproliferative effects of these compounds is an eventual precursor to cell death. However, inhibition of cell proliferation may be a more sensitive endpoint under the conditions employed because significant effects were observed for twice as many compounds on proliferation compared with viability. Thus, on the basis of this screen, one possible prioritization scheme would be to group these seven chemicals into the highest priority group. Diphenhydramine, which caused a 10% decrease in viability at the highest concentration tested would be given a lower priority than these first seven. The other eight compounds, which were generally without effect on either endpoint, would be the lowest priority for additional testing. For the present set of compounds, this approach would correctly group six of the eight compounds considered developmentally neurotoxic into the highest priority group, while including only one of the compounds considered nontoxic in this group. This information would be considered in the context of data from other screens (e.g., neurite outgrowth; Radio et al., in press), synaptogenesis, etc.) prior to making final prioritizations for testing in vivo. In this respect, the present assay would have identified lead as a high priority for additional testing, whereas this compound did not have effects on neurite outgrowth (Radio et al., in press), which is an example of the advantage of a battery of tests. By contrast, valproic acid and 5,5'-diphenylhydantoin were without effect in both the proliferation and the neurite outgrowth assays. It is possible that these compounds cause developmental neurotoxicity via other mechanisms that would be detected with assays on other endpoints. Alternatively, it is noteworthy that both of these compounds are therapeutic drugs, rather than environmental compounds. Thus, they are presumably more soluble, and perhaps effects would have been observed in either assay had higher concentrations been examined. Future experiments will consider this possibility.
Summary
The present work demonstrates the development of a novel screen that unites the assessment of chemical effects on cell proliferation, a critical process for neural development, with a high-throughput approach. The data demonstrate that ReNcell CX cells, an immortalized hNPC line, are an appropriate in vitro model of hNPCs for screening approaches. It should be noted that the present experiments did not attempt to calculate prediction ("false positive"/"false negative") rates for either prediction of effects on proliferation or developmental neurotoxicity. In either case, larger numbers of compounds need to be tested. Overall, the present data indicate that screening chemicals for effects on proliferation and viability in hNPCs should be considered for inclusion in a battery of tests to screen chemicals for the potential to cause developmental neurotoxicity (hazard identification)and prioritize them for additional testing.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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This work was funded by the U.S. Environmental Protection Agency. This document has been subjected to review by the National Health and Environmental Effects Research Laboratory and is approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. JMB was supported by the US EPA/UNC Toxicology Research Program, Training Agreement #CR8332370 with the Curriculum in Toxicology, University of North Carolina at Chapel Hill.
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1 Current address: Cellumen, Inc., Pittsburgh, PA.
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ACKNOWLEDGMENTS |
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We would like to thank Dr Stephanie Padilla and Dr Keith Houck for their critical reviews of the manuscript, and Theresa Freudenrich for technical support.
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