AN ADVANTAGE RESEARCH OF DEPLETED URANIUM & TUNGSTEN HEAVY ALLOY

Neurotoxic Potential of Depleted Uranium—Effects in Primary Cortical Neuron Cultures and in Caenorhabdit is elegans
ToxSci Advance Access originally published online on July 16, 2007
http://toxsci.oxfordjournals.org/cgi/content/full/99/2/553
Toxicological Sciences 2007 99(2):553-565; doi:10.1093/toxsci/kfm171

George C.-T. Jiang*, Kristen Tidwell , Beth Ann McLaughlin , Jiyang Cai , Ramesh C. Gupta , Dejan Milatovic¶, Richard Nass¶ and Michael Aschner¶,1
* Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1083 Department of Neurology Vanderbilt Eye Institute, Vanderbilt University, Nashville, Tennessee 37232 Toxicology Department, Murray State University, Hopkinsville, Kentucky 42240 ¶ Department of Pediatrics, Vanderbilt University, Nashville, Tennessee 37232
1 To whom correspondence should be addressed at Departments of Pediatrics and Pharmacology, and the Vanderbilt Kennedy Center for Research on Human Development, Vanderbilt University, 6110 MRBIII, 465 21st Ave. S, Nashville, TN 37232-2495. Fax: (615) 322-6541. E-mail: michael.aschner@vanderbilt.edu
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Received April 17, 2007; accepted June 13, 2007

ABSTRACTDepleted uranium (DU) is an extremely dense metal that is used in radiation shielding, counterbalances, armor, and ammunition. In light of the public concerns about exposure to DU and its potential role in Gulf War Syndrome (GWS), this study evaluated the neurotoxic potential of DU using focused studies on primary rat cortical neurons and the nematode Caenorhabditis elegans. We examined cell viability, cellular energy metabolism, thiol metabolite oxidation, and lipid peroxidation following exposure of cultured neurons to DU, in the form of uranyl acetate. We concurrently evaluated the neurotoxicity of uranyl acetate in C. elegans using various neuronal–green fluourescent protein reporter strains to visualize neurodegeneration. Our studies indicate that uranyl acetate has low cytotoxic potential, and uranium exposure does not result in significant changes in cellular energy metabolism, thiol metabolite oxidation, or lipid peroxidation. Furthermore, our C. elegans studies do not show any significant neurodegeneration following uranyl acetate exposure. Together, these studies suggest that DU, in the form of uranyl acetate, has low neurotoxic potential. These findings should alleviate the some of public concerns regarding DU as an etiologic agent of neurodegenerative conditions associated with GWS.
Key Words: depleted uranium; primary neurons; neurotoxicity; Gulf War Syndrome; C. elegans.

INTRODUCTIONDepleted uranium (DU) is a by-product of the enrichment of naturally occurring uranium for its most radioactive isotope, 235U. The extremely dense and pyrophoric properties of DU make it an excellent metallic substrate for radiation shielding, counterbalances, and in armor and ammunition (Jiang and Aschner, 2006 ). As a heavy metal, internalized DU is cleared by the kidneys, and numerous studies have demonstrated nephrotoxicity after exposure to high levels of DU (Andrews and Bates, 1987 ; Carriere et al., 2005 ; Goldman et al., 2006 ; Kobayashi et al., 1984 ; Taulan et al., 2004 ). Other than the effects on the kidneys, DU exposure is thought to result in neurologic sequelae. Indeed, it has been hypothesized that DU may contribute to the etiology of Gulf War Syndrome (GWS) (Abu-Qare and Abou-Donia, 2002 ; Bem and Bou-Rabee, 2004 ; Doucet, 1994 ; Durakovic, 2003 ; Gronseth, 2005 ; Jamal et al., 1996 ; Jiang and Aschner, 2006 ). Follow-up studies on Gulf War veterans exposed to DU demonstrated decreased cognitive performance compared to unexposed veterans, which provided evidence for such a theory (McDiarmid et al., 2000 ). The increased usage and health concerns have led researchers to scrutinize the effects of DU exposure on the central nervous system (CNS).
The recent interest in the effects of DU exposure on the CNS has led to a number of studies with small animals. Such studies have shown that uranium (U) indeed crosses the blood–brain barrier (Abou-Donia et al., 2002 ; Barber et al., 2005 ; Briner and Murray, 2005 ; Fitsanakis et al., 2006 ; Houpert et al., 2004 ; Leggett and Pellmar, 2003 ; Lestaevel et al., 2005 ; Paquet et al., 2006 ; Pellmar et al., 1999a ,b), accumulates in a dose-dependent manner in specific brain structures (Fitsanakis et al., 2006 ; Pellmar et al., 1999a ), and results in increased lipid oxidation (Briner and Murray, 2005 ), nitric oxide generation (Abou-Donia et al., 2002 ), and sensorimotor deficits (Abou-Donia et al., 2002 ). These studies have attempted to correlate the observed neurobiological changes with potential functional changes in cognitive behavior (Abou-Donia et al., 2002 ; Belles et al., 2005 ; Briner and Murray, 2005 ; Houpert et al., 2005 ). To date, however, there remains a significant gap in understanding the specific effects of uranium on cells of the CNS, and the potential molecular changes involved upon DU exposure.
The cellular effects of DU have only been evaluated in a limited number of cell culture models. Studies in Chinese hamster ovary cells have demonstrated cytogenetic toxicity of uranium (Lin et al., 1993 ), and induction of hypoxanthine (guanine) phosphoribosyltransferase (hprt) mutations and DNA adducts (Albertini et al., 2003 ; Stearns et al., 2005 ). Studies with immortalized human osteoblast cells to evaluate the effects of DU have corroborated this finding, further demonstrating that DU results in genotoxicity, and that it can be neoplastic (Miller et al., 1998a , 2001, 2002, 2003). Uranium has also been shown to induce activation of stress gene expression in human liver carcinoma cells (HepG2) (Miller et al., 2004 ). In the mouse macrophage cell line, J774, uranium treatment resulted in time- and concentration-dependent uptake of uranium, cytotoxicity, and induction of apoptosis (Kalinich et al., 2002 ). Concentration-dependent cytotoxicity was also observed in NRK-52E cells, another immortalized cell culture model representative of rat kidney proximal epithelium cells (Carriere et al., 2004 ). Researchers have also evaluated the transcriptomic and proteomic responses of HEK293 kidney cells, and renal tissue from rats exposed to DU, and found that there were several oxidative-response–related transcripts that were upregulated, and significantly increased peroxide levels that support the implication of oxidative stress (Prat et al., 2005 ; Taulan et al., 2004, 2006 ). In rat brain endothelial cells, the closest in vitro model to cells of CNS origin, researchers demonstrated that uranium did not result in significant cytotoxicity (Dobson et al., 2006 ).
To date, researchers have not undertaken focused studies to determine the effects of DU on cells of CNS origin. Numerous CNS cell models are available for study, including primary cultures and immortalized cell lines. Although primary cultures have a finite life span compared to immortalized cell lines, the former offer many advantages as cell lines will often show numerous changes in cell cycle and proliferation, morphology, and chromosomal variations. Furthermore, primary are cultured in the context of their naturally occurring neighboring cell types. In these studies, we have attempted to fill the gap in the knowledge of DU neurotoxicity by performing focused studies using primary rat cortical neurons to examine the acute neurotoxic potential of DU and the specific cellular effects in neurons. We are testing the hypothesis that DU results in significant concentration-dependent cytotoxicity, and oxidative stress, as has been previously seen in other cell culture models.
The nematode, Caenorhabditis elegans, is an excellent model organism that has been used in a number of toxicological studies (Anderson and Wild, 1994 ; Dhawan et al., 1999 ; Reichert and Menzel, 2005 ; Swain et al., 2004 ). The worms are easily grown and maintained, and have a rapid replication cycle, allowing for thousands of worms to be evaluated within a number of days (Brenner, 1974 ). The nematode is a model organism, with its complete genome determined, numerous genetic mutants freely available, and multicolor reporter constructs, e.g., green fluorescent protein (GFP), can be easily introduced into the system (Hobert and Loria, 2006 ; Link and Johnson, 2002 ; Miller, et al., 1999 ). Furthermore, there are only 302 neurons in the nematode, in which all the projection pathways have been determined (Gally and Bessereau, 2003 ; Wadsworth and Hedgecock, 1992 ). All of these C. elegans characteristics make it a powerful organism to evaluate the toxicological potential of a wide array of compounds. For our studies, C. elegans is an organism in which we can evaluate the in vivo effects of uranium on CNS cells. We tested the hypothesis that uranium exposure results in significant concentration-dependent neurotoxicity as can be visualized by neurodegeneration.
In light of the public concerns regarding DU, this study sought to evaluate the neurotoxicity of DU, in the form of uranyl acetate, using focused studies of a relatively homogeneous cell population of CNS origin. Here, we investigate the cytotoxic effects of U in primary rat neuronal cultures, subsequent changes in cellular metabolism, and concurrently evaluate the neurotoxicity of U in C. elegans using neuronal-GFP reporter strains.
MATERIALS AND METHODS

Materials
Uranyl acetate (UO2(CH3COO)2·2H2O) was purchased from Ted Pella, Inc. (Redding, CA). All other chemicals were purchased from Sigma (St Louis, MO) unless otherwise stated. Coverslips for cell culture were purchased from Carolina Biological Supply (Burlington, NC). All tissue culture media and supplements were purchased from Invitrogen (Carlsbad, CA), except for Hyclone Fetal Bovine Serum and Hyclone F12, which were purchased from VWR (Suwanee, GA). Nematode growth reagents and plasticware were purchased from VWR.
Cell culture conditions and uranyl acetate treatments.All experiments were approved by the Institutional Animal Care and Use Committee of Vanderbilt University and were performed according to Guidelines for Animal Experimentation as set forth by Vanderbilt University. Rat cortical neuron cultures were prepared from E17 rat pups, as previously described (McLaughlin et al., 1998 ). Briefly, E17 Harlan Sprague–Dawley rat embryos were decapitated, and the brains rapidly removed and placed in a 35-mm petri-dishes with cold Hank's balanced salt solution (HBSS). The cortices were dissected under a dissection microscope and then were placed in another dish containing HBSS to further remove blood vessels and meninges from cortical tissues. The isolated cortices were then transferred to a petri-dish containing 0.6% (wt/vol) trypsin in HBSS for 30 min. After two washes in HBSS, the cortical tissues were mechanically dissociated with a glass Pasteur pipette. Dissociated cortical cells were plated on poly-L-ornithine-treated glass coverslips in six-well plates, using a plating medium of glutamine-free Dulbecco's modified Eagle's medium–Eagle's salts (Invitrogen), supplemented with Ham's F12 (Hyclone, Logan, UT), heat-inactivated fetal bovine serum (Hyclone), and penicillin/streptomycin (Sigma), at a density of 700,000 cells per well. After 2 days in vitro, nonneuronal cell division was halted by a 1-day exposure to 10µM cytosine arabinoside (Sigma), and cultures were shifted to Neurobasal media (Invitrogen), supplemented with B27 (Invitrogen) and penicillin/streptomycin. Cells were maintained by changing the media every 2–3 days and grown at 37°C in a humidified atmosphere of 5% CO2 in air.
Cells were treated 3 weeks after isolation with DU (uranyl acetate), prepared as sterile solutions in treatment buffer, for 24 h, at 37°C in a humidified atmosphere of 5% CO2 in air. Treatment buffer consisted of minimal essential media (Invitrogen) supplemented with 25mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 10 ml N2 media supplement (Invitrogen), 0.001% BSA (Sigma). N-methyl-D-aspartate (NMDA, Sigma) was used as a positive control for cytotoxicity at a final concentration of 100µM in conjunction with 10µM glycine.
Cell viability determinations.Primary rat cortical neuron viability was determined by fluorescence activated cell sorting (FACS) using the LIVE/DEAD viability/cytotoxicity kit (Molecular Probes, Eugene, OR). Both floating and attached cells were collected and stained with 2 µl of calcein and 8 µl of ethidium homodimer in phosphate buffered saline (PBS) as previously described (Chen et al., 2002 ). The percentage of viable cells was analyzed by flow cytometry (BD Immunocytometry Systems, San Jose, CA). For each sample, at least 10,000 cells were counted on a BD FACScan (Becton Dickinson, San Jose, CA). Data analyses were performed with WinMDI (Windows Multiple Document Interafce for Flow Cytometry) (http://facs.scripps.edu).
Cell viability and proliferation were evaluated by lactate dehydrogenase (LDH) (Sigma) and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) (Sigma) assays. LDH release was measured with an in vitro toxicology assay kit (Sigma) by assaying 40 µl sample medium spectrophotometrically (490:630 nm) according to the manufacturer's protocol, to obtain a measure of cytoplasmic LDH released from dead and dying neurons (Legrand et al., 1992 ). MTT is yellow until reduced to purple formazan in the mitochondria of living cells. The reduction of MTT to formazan occurs only when mitochondrial reductase enzymes are active, and thus conversion is a measurement of mitochondrial inhibition, and can be correlated to the number of viable (living) cells. LDH release and MTT analyses were determined according to manufacturer's instructions. LDH release results were confirmed qualitatively by visual inspection of the cells and, in several instances, quantitatively by cell counts by the method of Rosenberg and Aizenman (1989) .
Thiol metabolite determination.Quantification of levels of glutathione (GSH) and its related products were performed by high-performance liquid chromatography (HPLC) as previously described (Jones, 2002 ; Jones et al., 1998 ; Nelson et al., 1999 ). Briefly, treated cells were washed with PBS, and resuspended in 0.5% perchloric acid with 0.2M boric acid and 10µM -Glu–Glu (internal standard), and sonicated with a Sonics Vibra-Cell, two times for 20 s at 25% power. Extracts were derivatized with iodoacetic acid and dansyl chloride. The acid soluble cysteine (Cys), cystine (CySS), GSH, and oxidized glutathione (GSSG) were analyzed by HPLC using fluorescence detection on a Waters 2695 Alliance HPLC system (Waters, Milford, MA). Samples were loaded onto an YMC Pack NH2 (amino) column (Waters) and were eluted with a gradient of sodium acetate. The solvent used for mobile phase was 80% methanol. The peaks were quantified by integration relative to the internal standard. Using this method, samples were analyzed for Cys, CySS, GSH, and GSSG content. Redox status for the GSH/GSSG redox couple (Eh GSH), and the Cys/CySS redox couple (Eh Cys) were calculated using the Nernst equation.
Total adenosine nucleotides determination.Changes in adenosine nucleotides were measured by isocratic reversed-phase HPLC as previously described (Yang et al., 2004 ). For HPLC analysis, treatment media was removed from the cell samples before adding 950 µl of chilled 0.3M perchloric acid with 1mM disodium ethylenediaminetetraacetate to each well to harvest cell extracts into microcentrifuge tubes. An aliquot of 2M potassium hydroxide (170 µl) was then added to each sample, followed by centrifugation at 9000 x g to remove precipitates of KClO4. The supernatant was then stored at – 80°C until HPLC analysis on a Waters HPLC system (Waters), coupled with a dual -absorbance UV detector (Model 2487) equipped to a computer system with Waters Millennium software program (Workstation v. 4.0) for data processing. The mobile phase used was 0.1M ammonium dihydrogen phosphate (pH 6.0) with 1% methanol. Using the Symmetry Shield C-18 column and a flow rate of 0.6 ml/min, the peaks of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were eluted at retention times of 3.462, 3.868, and 5.694 min, respectively, with a variation window of 0.2 min in both standard and sample extracts. The peak height responses for all three nucleotides were recorded at 206 nm. The concentration of each nucleotide was determined in a 15-µl sample extract injected to HPLC and finally expressed in terms of nmol nucleotide per ml extract. The total adenosine nucleotides (TAN) content was calculated by TAN = ATP + ADP + AMP, while the energy charge potential (ECP) was calculated by the equation ECP = [ATP + 0.5 (ADP)]/TAN, as previously described (Yang et al., 2004 ).
F2-IsoP quantitation.Quantification of F2-isoprostanes (F2-IsoP) levels was determined using a stable isotope dilution method with detection by gas chromatography/mass spectrometry and selective ion monitoring as previously described (Milatovic et al., 2005 ; Morrow and Roberts, 1991 ; Roberts and Morrow, 1994 ). Briefly, samples were extracted and saponified, a stable isotope internal standard added, and then prepared for gas chromatography through a series of purifications by C-18 and Silica Sep-Pak cartridges and thin layer chromatography (TLC). Gas chromatography was performed using a 15 m long, 0.25 mm diameter, 0.25-µm film thickness, DB1701 fused silica capillary column (Fisons, Folsom, CA). The injector temperature was 265°C and oven (column) temperature was programmed from 200°C to 300°C at 15°C/min. Helium was used as the carrier gas at a flow rate of 1 ml/min. Ion source temperature was 250°C, electron energy was 70 eV, and filament current was 0.25 mA. For analysis, compounds were dissolved in undecane that was dried over a bed of calcium hydride. Negative ion chemical ionization mass spectrometry was performed using an Agilent Technologies G1789A GC/MSD instrument with a Hewlett–Packard computer system with ChemStation-NT. Total protein content was determined by BCA assay (Pierce, Rockford, IL) with bovine serum albumin as the standard (Smith et al., 1985 ).
Strains and maintenance.Caenorhabditis elegans strains were cultured on bacterial lawns of either NA-22 or OP-50, seeded on 8P or nematode growth medium (NGM) plates respectively, at 20°C according to standard methods (Brenner, 1974 ). Caenorhabditis elegans strain N2 (var. Bristol) is the wild-type strain, and was a gift of Dr Richard Nass (Vanderbilt University, Nashville, TN). The BY250 strain was developed and obtained from Dr Richard Nass (Vanderbilt University, Nashville, TN). Strain NW1229 (dpy-20(e1362) IV; evIs111) was obtained from the C. elegans Genetics Center (CGC, University of Minnesota, Minneapolis, MN).
Exposure of C. elegans to uranyl acetate.Embryos were obtained by hypochlorite treatment of gravid adults (Lewis and Fleming, 1995 ). After 17–24 h incubation in M9 buffer to obtain synchronized L1s, such that all nematodes are at the same point in their life cycle, the worms were washed once in 10 ml of dH2O, and then diluted to 50 worms per µl. L1 worms were treated with DU (uranyl acetate), prepared from a 1M stock solution in water. Five thousand worms were used in each siliconized microcentrifuge tube (Denver Scientific Inc., Metuchen, NJ) per treatment assay, and incubated with gentle shaking at 800 rpm for 30 min on a VWR Digital Mini Vortex Mixer (VWR Scientific, Suwanee, GA). Worms were then spread on NGM/OP-50 plates and incubated for 24 h at 20°C before further evaluation. For quantitative analyses of uranyl acetate-induced changes in worm viability, total number of live worms was determined for each concentration by counting each plate under a Stemi-2000 dissecting microscope (Zeiss, Thornwood, NY).
Photomicroscopy.Cell morphology was visually inspected on a Zeiss Axiovert 40 inverted microscope (Zeiss, Thornwood, NY). Cortical culture images were captured on an inverted Nomarski microscope (Zeiss Axiovert 200M) with AxioCam and AxioVision 4.4 software (Zeiss), using fixed exposures for all image captures between different treatments. Nematode images were captured on a Zeiss upright LSM510 confocal microscope (Zeiss), using laser scanning fluorescence and DIC (Nomarski) imaging. Worms were photographed under oil immersion with a 40x/1.30 Plan-Neofluar objective using fixed exposure settings for all image captures between different treatments. Images were exported using the Zeiss LSM Image Browser. Images were quantified for their fluorescence using Adobe Photoshop 6.0 (Adobe, San Jose, CA) and NIH ImageJ software. The fluorescent intensities were subsequently used to test if the levels of fluorescence were decreased upon treatment with U. With BY250 worms, cell bodies and dendrites were also manually scored as present if fluorescence could be seen. Dendrites were scored as abnormal if they had breaks or were barely visible. The ratio of abnormal:normal dendrites was used to calculate ratios for the different treatments, which were then compared for significance as previously described (Nass and Blakely, 2003 ).
Data analysis।All results are given as mean ± standard error of the mean. Differences between groups were analyzed statistically with one-way ANOVA followed by post hoc tests for multiple comparisons with p <>



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