ToxSci Advance Access originally published online on April 27, 2007
Toxicological Sciences 2007 98(1):198-205; doi:10.1093/toxsci/kfm095
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Manganese Induces Oxidative Impairment in Cultured Rat Astrocytes
,1
* Departments of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
Murray State University, Breathitt Veterinary Center, Toxicology Department, Hopkinsville, Kentucky 42241
Department of Neurotoxicology, Medical Research Centre, Polish Academy of Sciences, 02-792 Warsaw, Poland
Pharmacology, and the Kennedy Center for Research on Human Development, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
1 To whom correspondence should be addressed at Department of Pediatrics, B-3307 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232-2495. Fax: (615) 322-6541. E-mail: Michael.Aschner{at}vanderbilt.edu.
Received February 21, 2007; accepted April 18, 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Excessive free radical formation has been implicated as a causative factor in neurotoxic damage associated with exposures to a variety of metals, including manganese (Mn). It is well established that Mn accumulates in astrocytes, affecting their ability to indirectly induce and/or exacerbate neuronal dysfunction. The present study examined the effects of Mn treatment on the following endpoints in primary astrocyte cultures: (1) oxidative injury, (2) alterations in high-energy phosphate (adenosine 5'-triphosphate, ATP) levels, (3) mitochondrial inner membrane potential, and (4) glutamine uptake and the expression of glutamine transporters. We quantified astrocyte cerebral oxidative damage by measuring F2-isoprostanes (F2-IsoPs) using stable isotope dilution methods followed by gas chromatography–mass spectrometry with selective ion monitoring. Our data showed a significant (p < 0.01) elevation in F2-IsoPs levels at 2 h following exposure to Mn (100µM, 500µM, or 1mM). Consistent with this observation, Mn induced a concentration-dependent reduction in ATP and the inner mitochondrial membrane potential (
m), measured by the high pressure liquid chromatography method and the potentiometric dye, tetramethyl rhodamine ethyl ester, respectively. Moreover, 30 min of pretreatment with Mn (100µM, 500µM, or 1mM) inhibited the net uptake of glutamine (GLN) (3H-glutamine) measured at 1 and 5 min. Expression of the messenger RNA coding the GLN transporters, SNAT3/SN1 and SNAT1, was inhibited after 100 and 500µM Mn treatment for 24 h. Our results demonstrate that induction of oxidative stress, associated mitochondrial dysfunction, and alterations in GLN/glutamate cycling in astrocytes represent key mechanisms by which Mn exerts its neurotoxicity.
Key Words: astrocyte; manganese; F2-isoprostanes; mitochondria; ATP; m; glutamine.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Manganese (Mn) is a naturally occurring trace metal commonly found in the environment. It is essential for maintaining the proper function and regulation of many biochemical and cellular reactions (Takeda, 2003
). Despite its essentiality, Mn is a common environmental contaminant, which can exert neurotoxic activity in human cells. Major sources of environmental contamination with Mn include the manufacturing of alloys, steel, iron, and fertilizers, as well as mining operations (Aschner, 2000). Excessive accumulation of Mn in specific brain areas, the substantia nigra, the globus pallidus (GP), and the striatum, produce neurotoxicity resulting in a degenerative brain disorder, referred to as manganism. It is characterized by clinical signs and morphological lesions similar to those seen in Parkinson's disease (PD) (Racette et al., 2001). In addition to targeting similar brain areas, PD and manganism share multiple common mechanisms leading to dopaminergic neurodegeneration, namely, mitochondrial dysfunction, aberrant signal transduction, oxidative stress, protein aggregation, and the activation of cell death pathways (Dobson et al., 2004; HaMai and Bondy, 2004; Kitazawa et al., 2005; Latchoumycandane et al., 2005).
At the cellular level, Mn preferentially accumulates in mi-tochondria, where it disrupts oxidative phosphorylation and increases the generation of reactive oxygen species (ROS) (Gunter et al., 2006). Excessive production of ROS induces the oxidation of membrane polyunsaturated fatty acids, yielding a multitude of lipid peroxidation products. One such family of products is the F2-isoprostanes (F2-IsoPs), prostaglandin-like molecules produced by free radical–mediated peroxidation of arachidonic acid (Morrow and Roberts, 1999). These biomarkers of oxidative stress have been investigated in several in vivo models associated with free radical damage, but not within the context of Mn-induced neurotoxicity. In addition, ROS generation, the ensuing decrease in mitochondrial membrane potential, and the depletion of high-energy phosphates affect mitochondrial permeability transition (MPT), leading to organelle swelling, disruption of the outer membrane, and release of numerous apoptogenic factors into the cytosol (Green and Reed, 1998).
Given the propensity of astrocytes to accumulate Mn, it is reasonable to postulate that they represent an initial site of Mn-induced damage. Astrocytes perform functions essential for normal neuronal activity, including glutamate (GLU) uptake (80% of synaptic GLU), glutamine (GLN) release, K+ and H+ buffering and water transport (Kimelberg and Aschner, 1998). A principal neuromodulatory role of astrocytes is the control over GLU–GLN homeostasis. GLU is metabolized to GLN by the astrocyte-specific enzyme, glutamine synthase (GS). This GLU–GLN pathway constitutes the source of a GLU pool in the brain. GS contains eight Mn ions per octamer and accounts for approximately 80% of the total endogenous Mn in the brain. Unlike neurons, astrocytes have the ability to concentrate Mn to levels 50-fold higher than the culture media (Aschner et al., 1992); this provides a mechanism by which Mn concentrations in the astrocytic cytosol attain the range required for optimal activation of GS, thereby ensuring optimal production of GLN for subsequent neuronal uptake and metabolism into 
-aminobutyric acid (GABA). However, excessive Mn uptake and subsequent sequestration by mitochondria may result in cellular dysfunction as discussed above.
GLN efflux from astrocytes is mediated by the sodium-coupled neutral amino acid transporter 3 (SNAT3, SN1), a System N amino acid transporter localized to perisynaptic astrocytes, which specifically transports only GLN, histidine, and asparagine (Chaudry et al., 2002). System N is a Na+-glutamine symporter and an H+ antiporter. Another GLN-accepting amino acid transporter more abundantly expressed in astrocytes than in neurons is ASCT2 (Dolinska et al., 2004). System ASC is a sodium-dependent antiporter which exists as two different isoforms termed ASCT1 and ASCT2, with ASCT2 having an affinity for GLN. GLN uptake in neurons is also mediated by sodium-dependent transporters of the System A family, two of which, SNAT1 (GlnT, SAT1, ATA1, SA2) and SNAT2 (SAT2, ATA2), are known to modulate GLU and/or GABA recycling, and, thereby, synaptic function (Heckel et al., 2003). A previous study from our laboratory has shown that exposure of astrocytes to acrylamide or methylmercury affects astrocytic GLN uptake and expression of messenger RNA (mRNA) encoding for GLN transporters (Aschner et al., 2005; Wu et al., 2005; Yin et al., 2007). Furthermore, excessive accumulation of GLN and alterations in GLN recycling, as a result of interference with mitochondrial function, are deleterious to brain function (Albrecht et al., 2007).
In view of the essential function of astrocytes for maintaining control over the composition of the extracellular fluid and normal neuronal activity, we evaluated Mn's effects on oxidative stress; alterations in adenosine 5'-triphosphate (ATP) levels; mitochondrial inner membrane potential (m); GLN uptake; and expression of SNAT3, ASCT2, and SNAT1 mRNA.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Materials.
L-[G-3H]-GLN (specific activity: 49.0 Ci/mmol) was purchased from Amersham Biosciences (Piscataway, NJ). Manganese chloride (MnCl2) and ATP standards were purchased from Sigma Chemical Co. (St Louis, MO). Minimal essential medium (MEM) with Earle's salts, heat-inactivated horse serum, penicillin, streptomycin, and tetramethyl rodamine ethyl ester (TMRE) were purchased from Invitrogen (Carlsbad, CA). 4H2-labeled 15-F2
-IsoP internal standard and prostaglandin F2 methyl ester were purchased from Cayman chemicals (Ann Arbor, MI).
Primary astrocytes culture.
The procedure for isolation and culturing of astrocytes from cerebral cortices of newborn (1 day old) Sprague–Dawley rats (Aschner et al., 1994) was approved by the institutional committee for animal research and was performed in accordance with the guidelines of the National Institutes of Health (NIH) for the care and use of laboratory animals. Briefly, rat pups were decapitated, and the cerebral cortices were removed. After removal of the meninges, the cerebral cortices were digested with bacterial neutral protease (dispase, Invitrogen), and astrocytes were recovered by repeated removal of dissociated cells from brain tissues. Twenty-four hours after the initial plating in 6- and 12-well plates, the media were changed to preserve the adhering astrocytes and to remove neurons and oligodendrocytes. The cultures were maintained at 37°C in a 95% air/5% CO2 incubator for 3–4 weeks in MEM with Earle's salts supplemented with 10% heat-inactivated horse serum, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. The media were changed twice per week. The surface-adhering monolayer cultures were > 95% positive for the astrocytic marker, glial fibrillary acidic protein.
F2-IsoPs assay.
Total F2-IsoPs were measured in primary astrocyte cultures exposed to 100µM, 500µM, or 1mM of MnCl2 for 30 min, 2 or 6 h by using gas chromatography/mass spectrometry with selective ion monitoring (Morrow and Roberts, 1999). Briefly, cells were resuspended in 0.5 ml of methanol containing 0.005% butylated hydroxytoluene, sonicated, and then subjected to chemical saponification using 15% KOH to hydrolyze bound F2-IsoPs. The cell lysates were adjusted to a pH of 3, followed by the addition of 0.1 ng of 4H2-labeled 15-F2-IsoP internal standard. F2-IsoPs were subsequently purified by C18 and silica Sep-Pak extraction and by thin layer chromatography. They were then analyzed by pentafluorobenzyl ester, a trimethylsilyl ether derivative, via gas chromatography, negative ion chemical ionization mass spectrometry.
Mn concentrations in this study (100µM, 500µM, or 1mM) were determined based on the relevant toxic Mn effect on mammalian cells as described in the literature. For example, weekly injections of Mn over a 3-month period (0, 2.25, 4.5, or 9 g) in the striatum and the GP of monkeys have been shown to produce dose-related clinical signs, which are more severe in the higher dose ranges (Suzuki et al., 1975). At the highest dose, the Mn concentration is increased 12-fold in the striatum and ninefold in the GP. The "physiological range" (no symptoms) is 75–100µM, and clinical signs increase in frequency and severity above this level, suggesting that this concentration is near the threshold of toxicity.
Measurement of ATP.
Levels of ATP were analyzed by an isocratic reversed-phase high pressure liquid chromatography (HPLC) method (Yang et al., 2004). ATP was extracted from control and Mn-exposed astrocytes by adding 950 µl of ice-cold perchloric acid (0.2M) containing Na-ehtylenediaminetetraacetic acid (1mM) to the primary astrocyte culture plates immediately after the medium was removed. The cells were then scraped off the bottom of the plates, and the acid extract was transferred to a microcentrifuge tube. The acid extract was neutralized with 170 µl of potassium hydroxide (KOH; 2M) and centrifuged at 9000 x g for 5 min to remove fine precipitates of perchlorate (KCLO4). The supernatants were stored at –20°C before being subjected to ATP determination. The concentration of ATP was determined in a 15 µl of sample extract injected into HPLC with ultraviolet detector and 0.1M of ammonium dihydrogen phosphate (pH 6.0) containing 1% ethanol as a mobile phase. Using the Symmetry Shield C-18 column and a flow rate of 0.6 min/ml, the peak of ATP production was eluted at a retention time of 3.462 min and recorded at 206 nm.
Measurement of mitochondrial membrane potential (m).
The m was measured with the fluorescent dye, TMRE (Rao and Norenberg, 2004). At the end of the treatments with 100µM, 500µM, or 1mM of Mn for 1 or 6 h, the culture medium was removed (duplicate plates per experiment; repeated three times using different astrocyte isolations), and the cells were loaded with TMRE at a final concentration of 50nM in N-2-hydroxy-ethylpiperazine N'-2-ethansulfonic acid (HEPES) buffer for 20 min at 37°C in a 5% CO2 incubator. Cells were rinsed with phosphate-buffered saline and examined with a Zeiss inverted fluorescent microscope (Zeiss Axiovert S100, Carl Zeiss MicroImaging Inc., Thornwood, NY) equipped with a cooled digital camera (Photometrics CoolSNAP, Roper Scientific Photometrics, Tucson, AZ). Images of various fields in each plate were captured at x10 magnification with the digital camera. Fluorescent intensities were calculated in eight randomly selected fields per experiment and were analyzed using the NIH software (Scion Incorporation, Frederick, MD). In each image field, the total number of pixels was quantified on a gray scale (0–255 counts), and the mean pixel value in each image field was expressed as mean ± SEM. The fluorescent intensities were expressed as percent fluorescence change from control; each experiment was performed in duplicate plates and repeated three times in astrocytes derived from independent isolations.
Analysis of 3H-glutamine uptake in astrocytes.
3H-glutamine uptake was assayed as previously described (Allen et al., 2001). Astrocytes were studied at 3 weeks when the monolayers were confluent. Cells in six-well plates were washed three times with 2 ml of fresh sodium-HEPES buffer consisting of the following: 122mM of NaCl, 3.3mM of KCl, 0.4mM of MgSO4, 1.3mM of CaCl2, 1.2mM of KH2PO4, 10mM of glucose, and 25mM of HEPES adjusted to a pH of 7.4 with 10M of NaOH. In preincubation experiments, cells were pretreated in Na-HEPES buffer only, or with Na-HEPES buffer containing Mn (100µM, 500µM, or 1mM) for 30 min in a 37°C, 95% air/5% CO2 incubator. Cells were then washed three times with 2 ml of Na-HEPES buffer. Thereafter, 1 ml of prewarmed buffer containing 1 µCi/ml of L-[G-3H]-GLN in the presence of unlabeled GLN (at a final concentration of 50µM) was added to each well, and GLN uptake was measured at 1 and 5 min at room temperature. At each time point, the reactions were terminated by aspirating the buffer from the well, followed by five washes with 2 ml of ice-cold mannitol buffer (290mM of mannitol, 10mM of Tris-nitrate, 0.5mM of Ca(NO3)2; pH adjusted to 7.4 with KOH). At the end of the experiment, cells were lysed in 2 ml of 1M NaOH. An aliquot of 25 µl was used for protein determination by the bicinchoninic acid protein assay (Pierce, Rockford, IL). An aliquot of 750 µl was used for radioactivity measurement. Uptake of GLN was expressed as cpm/mg of protein.
Reverse transcription-polymerase chain reaction.
Total RNA from cell cultures exposed to Mn concentration of 100µM, 500µM, or 1mM for 24 h was extracted using Trizol (Invitrogen). RNA (2 µg) was transcribed using Superscript II (Invitrogen). The primers were obtained from the Laboratory of DNA Sequencing, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw. Each complementary DNA (2 µl) was amplified by PCR using the primers for rat amino acid transporters: SNAT3, SNAT1, and ASCT2. The expression was quantitatively related to the gene coding for the constitutive protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequence of primers and the lengths of the products are outlined in Table 1. After 30 cycles of amplification (1 min at 9°C, 1 min at 59°C for SNAT3, and 55°C for ASCT2 and SNAT1 and 1 min at 72°C using Biometra Thermocycler), the PCR products were recorded using the Nucleovision system (Nucleotech, San Mateo, CA), and densitometric analysis was carried out using the GelExpert 4.0 program.
|
Statistical analysis.
Measurements of F2-IsoPs, ATP, and mitochondrial membrane potential, as well as experiments with 3H-glutamine uptake were conducted in duplicate or triplicate wells/experiment, and the mean from three to four independent experiments was used for statistical analysis. The data were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test with statistical significance set at p < 0.05. All analyses were carried out with GraphPad Prism 4.02 for Windows (GraphPad Software, San Diego, CA).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Effects of Mn on F2-IsoPs Formation in Cultured Astrocytes
We tested the ability of Mn to induce oxidative stress in primary astrocyte cultures by measuring levels of F2-IsoPs, a lipid peroxidation biomarker of oxidative injury. Neonatal rat primary astrocytes exposed to 500µM or 1mM of MnCl2 for 30 min, 2 or 6 h showed significant increases in F2-isoPs levels compared to controls (Fig. 1). Astrocyte cultures exposed to 1mM of Mn for 30 min showed a fivefold increase in F2-IsoPs compared to the control level (137.3 ± 4.3 pg/mg protein). The lowest MnCl2 concentration (100µM) induced a significant increase in markers of oxidative stress at 2 h, but not at 30 min or 6 h following Mn exposure.
|
Characterization of Cellular ATP Following Mn Exposure
Total cellular ATP levels were measured in MnCl2-exposed astrocyte cultures as an additional indicator of overt cellular toxicity and mitochondrial dysfunction. ATP levels were significantly decreased in astrocytes exposed for 30 min to the full range of investigated Mn concentrations (Fig. 2). The greatest depletion of ATP (63%) was seen in astrocytes exposed for 30 min to 1mM of MnCl2. Astrocyte cultures exposed for 30 min or 2 h to 500µM of Mn showed similar levels of ATP depletion to those exposed for 30 min or 2 h to only 1mM of Mn. In contrast, astrocytes exposed for 2 h to the lowest concentrations of Mn (100µM) did not show significant reductions in ATP levels.
|
Effects of Mn on the
m in Cultured AstrocytesThe
m, an indicator of mitochondrial dysfunction, was evaluated in astrocytes exposed to Mn. Treatment of astrocytes with 100µM, 500µM, or 1mM of Mn for 1 or 6 h resulted in significant dissipation of the m, as demonstrated by decreased mitochondrial TMRE fluorescence (Figs. 3A and 3C). Quantification of TMRE fluorescence intensities revealed that all the concentrations of Mn investigated induced significant dissipation of the m following 1 or 6 h of exposure (Figs. 3B and 3D). Fluorescence intensities of astrocytes exposed to 100µM, 500µM, or 1mM of Mn for 1 h were indistinguishable from intensities of astrocytes exposed to the same Mn concentrations for 6 h.
|
GLN Uptake Inhibition by Mn in Cultured Astrocytes
Compared with control uptake rates (100%), pretreatment of astrocytes for 30 min with Mn inhibited the net uptake of GLN in a concentration-dependent manner. All Mn treatments significantly inhibited GLN astrocytic uptake at 1 min and 5 min (Fig. 4). Furthermore, 1mM of Mn treatment induced greater inhibition of GLN uptake at 1 min compared to the inhibition induced at 5 min.
|
Influence of Mn on Expression of SNAT3, ASCT2, and SNAT1 mRNA
To explore molecular mechanisms associated with the effects of Mn on GLN uptake, we measured astrocytic mRNA expression of amino acid transporters by reverse transcription-PCR (RT-PCR). Figure 5 depicts the effects of Mn treatment on the mRNA expression of SNAT3, ASCT2, and SNAT1. The bars display the ratios of SNAT3, ASCT2, and SNAT1 to GAPDH, a constitutively expressed marker. Treatments with 100 and 500µM of Mn for 24 h significantly reduced the mRNA expression of SNAT3 and SNAT1, but did not reduce the mRNA expression of ASCT2 (Fig. 5).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The present study provides a direct link between mitochondrial dysfunction and oxidative stress due to Mn neurotoxicity. To our knowledge, this is the first study to investigate Mn-induced neurotoxicity in astrocytes by quantifying biomarkers of oxidative damage (F2-IsoPs) and energy metabolites (ATP). Our results show that increased lipid peroxidation, depletion of high-energy phosphates, and the collapse of the mitochondrial inner membrane potential (
m) represent early events and key mechanisms in Mn-induced astrocytic dysfunction.
We investigated Mn-induced oxidative stress in astrocytes by measuring F2-IsoPs (Morrow and Roberts, 1999), a group of arachidonic acid-derived prostanoid isomers generated by free radical damage due to arachidonic acid that are not extensively metabolized in situ. Moreover, the National Institute of Environmental Health Science Biomarkers Oxidative Stress Study (Kadiiska et al., 2005) recently proposed that F2-IsoPs are the most reliable biomarkers of oxidative damage. Astrocytes exposed to Mn concentrations known to elicit neurotoxic effects (500µM or 1mM), showed significant elevations in F2-IsoPs levels at all investigated exposure times (Fig. 1). Thus, increases in ROS, which are generated by electron leak from the electron transport chain (ETC) (Turrens and Boveris, 1980), potentially damaging mitochondria directly or through the effects of secondary oxidants like superoxide, H2O2, or peroxynitrite (ONOO–), mediate Mn-induced oxidative damage. Moreover, superoxide produced in the mitochondrial ETC may catalyze the transition shift of Mn2+ to Mn3+ through a set of reactions similar to those mediated by superoxide dismutase and thus lead to the increased oxidant capacity of this metal (Gunter et al., 2006). Consequent oxidative damage produces an array of deleterious effects: it may cause structural and functional derangement of the phospholipids bilayer of membranes, disrupt energy metabolism, metabolite biosynthesis, calcium and iron homeostasis, and initiate apoptosis (Attardi and Schatz, 1988; Uchida, 2003; Yang et al., 1997).
Interestingly, following exposure to a low MnCl2 concentration (100µM), levels of F2-IsoPs were significantly increased only at 2 h but were indistinguishable from controls (137.3 ± 4.3 pg/mg protein) at 30 min and 6 h (Fig. 1). A similar response of F2-IsoPs increase followed by full recovery was observed in our in vivo models of innate immunity- and excitotoxicity-mediated cerebral oxidative damage (Milatovic et al., 2003, 2005). F2-IsoPs were not significantly increased in the cerebra of mice following the intracerebroventricular (icv) injection of lipopolysaccharide (LPS, 5 µg in 5 µl of saline) at 10 h but were significantly increased at 24-h postinjection (Milatovic et al., 2003). By 72-h postinjection, F2-IsoPs values in LPS-treated mice had returned to baseline. Thus, oxidative stress has been implicated not only in metal-induced neurotoxicity (Dobson et al., 2004), but also in models of inflammation and excitotoxicity, as well as in a wide range of neurodegenerative conditions, such as PD and Alzheimer's disease (Albers and Beal, 2000). A previous report showed that neurons exposed to fuel additive, methylcyclopentadienyl manganese tricarbonyl, or MnCl2 are susceptible to oxidative stress, energy failure associated with mitochondrial dysfunction (Zwingmann et al., 2003), and mitochondria-induced apoptosis (Anantharam et al., 2002). Thus, it is likely that the oxidative impairment of astrocytic functions may indirectly induce and/or exacerbate neuronal dysfunction.
Consistent, and preceding the Mn-induced increased on F2-IsoPs levels (Fig. 1), we noted an early decrease in astrocytic ATP levels. Higher concentrations of MnCl2 (500µM and 1mM) induced significant (p < 0.01) decreases in ATP levels at all investigated exposure times (Fig. 2). As a consequence, ATP depletion or a perturbation in energy metabolism might diminish the ATP-requiring neuroprotective action of astrocytes, such as GLU and GLN uptake and free radical scavenging (Rao et al., 2001). In addition, depletion of high-energy phosphates may affect intracellular Ca2+ in astrocytes through mechanisms involving the disruption of mitochondrial Ca2+ signaling. This assertion is supported by data that clearly show that Mn inhibits Na+-dependent Ca2+ efflux (Gavin et al., 1990) and respiration in brain mitochondria (Zhang et al., 2004), both critical to maintaining normal ATP levels and ensuring adequate intermitochondrial signaling.
We further evaluated Mn-induced cytotoxicity in astrocytes by measuring lactate dehydrogenase (LDH) release from injured cells into the culture medium, as well as mitochondrial function, using 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The studies revealed that Mn exposure induced time- and concentration-dependent increase of LDH release and reduction of MTT. Astrocytes exposed to 1mM Mn for 2 h showed more than twofold increase in LDH and 30% decrease in MTT compared to controls.
Another consequence of increased oxidative stress and mitochondrial energy failure is the induction of the MPT, a Ca2+-dependent process characterized by the opening of the permeability transition pore in the inner mitochondrial membrane. This process results in increased permeability to protons, ions, and other solutes (Zoratti and Szabo, 1995), which, consequently, leads to a collapse of the mitochondrial inner membrane potential (m). Loss of the m results in colloid osmotic swelling of the mitochondria matrix, movement of metabolites across the inner membrane, defective oxidative phosphorylation, cessation of ATP synthesis, and further generation of ROS. Our experiments demonstrated a concentration-dependent effect of Mn on the mitochondrial inner membrane potential in cultured astrocytes (Fig. 3). This effect was noted already at 1-h postexposure to Mn, consistent with the reduction in ATP (Fig. 2) and increased production of F2-IsoPs (Fig. 1). Additional experiments in which we evaluated fluorescence intensities of astrocytes exposed to 500µM of Mn for 30 min demonstrated that dissipation of the m was indistinguishable between 30 min and 1 h exposure. Studies by Zhang et al. (2004) have shown that high levels of MnCl2 (1mM) cause a significant dissipation of the m in isolated rat brain mitochondria, consistent with induction of the MPT. Our results are also in agreement with an earlier study by Rao and Norenberg (2004), which found that Mn causes a dissipation of the m in a concentration- and time-dependent manner in astrocytes. The mechanism(s) by which Mn induces the MPT in astrocytes is not completely understood; however, increased production of ROS and associated oxidative stress are generally considered major factors of MPT induction (Castilho et al., 1995; Rao and Norenberg, 2004).
Our results show that pretreatment of astrocytes for 30 min with Mn inhibits the initial net uptake of GLN at 1 and 5 min in a concentration-dependent manner (Fig. 4). These observations are consistent with our findings that Mn decreases intracellular ATP as early as 30 min after exposure (Fig. 2). While one could certainly extend the GLN uptake studies to hours, we believe that the initial rates of uptake can be masked under those conditions, because it is hard to differentiate between frank uptake effects of Mn versus those on GLN efflux. As several of the GLN transporters are sodium-dependent (systems A, ASC, and N), dissipation of ionic gradients across membranes and the intracellular accumulation of sodium are consistent with reduced uptake of GLN in Mn-treated astrocytes. GLN is an important amino acid that plays a pivotal role in neuron–glia interactions, particularly in the turnover of the transmitter pool of GLU and GABA, the principal CNS excitatory and inhibitory neurotransmitters (Conti and Weinberg, 1999). Since neurons are not capable of anaplerosis, it is essential that they obtain precursors from astrocytes for transmitter synthesis. Glutamatergic and GABAergic neurons use glial GLN as precursors for neurotransmitter synthesis as a part of the GLU–GLN cycle (Sonnewald et al., 1997). After uptake by astrocytes, GLU can either be converted to glutamine directly by glutamine synthetase, or can enter the tricarboxylic acid cycle after conversion to 2-oxoglutarate for energy production and/or the synthesis of other metabolites. Inhibition of GLN uptake in Mn-treated astrocytes is, therefore, likely to reflect the decreased ability of cells for maintaining neuronal GLN and GABA pools as well as the generation of releasable GLN and GABA (Albrecht et al., 2007). In addition, our data also demonstrate that the inhibition of GLN uptake in Mn-treated astrocytes is accompanied by the inhibition of the expression of mRNA coding for SNAT3 and SNAT1 (Fig. 5). The GLN transport proteins, SNAT3 and SNAT1, are capable of mediating outward and inward transport of GLN, with the direction depending on the GLN and/or pH gradients (Chaudry et al., 2002). While cellular ATP levels are critical to Mn-induced GLN uptake kinetic, our results implicate impaired GLN shuttling between astrocytes and neurons as one of the possible causes of the derangement of amino acidergic neurotransmission known to be associated with Mn toxicity.
In summary, our studies demonstrate that the mechanisms of Mn-induced neurotoxicity are associated with the induction of oxidative stress, mitochondrial dysfunction, and alterations in GLU–GLN cycling. Once inside the astrocytes, Mn induced the effects of transient increases in ROS generation, simultaneous collapse of the mitochondrial membrane potential, and exacerbates the effects of existing defects in electron transport to slow ATP production. Releases of ROS mediate lipid peroxidation, which, consequently, diminish membrane permeability and affect GLN shuttling. Finally, we propose that oxidative stress generated through mitochondrial perturbation plays a key role in Mn-induced astrocytic dysfunction.
|
ACKNOWLEDGMENTS |
|---|
This study was supported by Public Health Service Grants ES10563 (M.A.) and ES013730 (J.L.A.) from the NIH and Department of Defense W81XWH-05-1-0239 (M.A.).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Albers DS, Beal MF. Mitochondrial dysfunction and oxidative stress in aging and neurodegenerative disease. J. Neural. Transm. Suppl. (2000) 59:133–154.[Medline]
Albrecht J, Sonnewald U, Waagepetersen HS, Schousboe A. Glutamine in the central nervous system: Function and dysfunction. Front. Biosci. (2007) 12:332–343.[CrossRef][Web of Science][Medline]
Allen JW, El-Oqayli H, Aschner M, Syversen T, Sonnewald U. Methylmercury has a selective effect on mitochondria in cultured astrocytes in the presence of [U-13C]glutamate. Brain Res. (2001) 908:149–154.[CrossRef][Web of Science][Medline]
Anantharam V, Kitazawa M, Wagner J, Kaul S, Kanthasamy AG. Caspase-3-dependent proteolytic cleavage of protein kinase C
is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl. J. Neurosci. (2002) 22:1738–1751.
Aschner M. Manganese in health and disease: From transport to neurotoxicity. Handbook of Neurotoxicology—Massaro E, ed. (2000) Totowa, NJ: Humana Press. 195–209.
Aschner M, Gannon M, Kimelberg HK. Manganese uptake and efflux in cultured rat astrocytes. J. Neurochem. (1992) 58:730–735.[Web of Science][Medline]
Aschner M, Mullaney KJ, Wagoner D, Lash LH, Kimelberg HK. Intracellular glutathione (GSH) levels modulate mercuric chloride (MC)- and methylmercuric chloride (MeHgCl)-induced amino acid release from neonatal rat primary astrocyte cultures. Brain Res. (1994) 664:133–140.[CrossRef][Web of Science][Medline]
Aschner M, Wu Q, Friedman MA. Effects of acrylamide on primary neonatal rat astrocyte functions. Ann. N. Y. Acad. Sci. (2005) 1053:444–454.[CrossRef][Web of Science][Medline]
Attardi G, Schatz G. Biogenesis of mitochondria. Annu. Rev. Cell Biol. (1988) 4:289–333.[CrossRef][Web of Science][Medline]
Castilho RF, Kowaltowski AJ, Meinicke AR, Bechara EJ, Vercesi AE. Permeabilization of the inner mitochondrial membrane by Ca2+ ions is stimulated by t-butyl hydroperoxide and mediated by reactive oxygen species generated by mitochondria. Free Radic. Biol. Med. (1995) 18:479–486.[CrossRef][Web of Science][Medline]
Chaudry FA, Reimer RJ, Edwards RH. The glutamine commute: Take the N line and transfer to the A. J. Cell Biol. (2002) 157:349–355.
Conti F, Weinberg RJ. Shaping at glutamatergic synapses. Trends Neurosci. (1999) 22:451–458.[CrossRef][Web of Science][Medline]
Dobson AW, Erikson KM, Aschner M. Manganese neurotoxicity. Ann. N. Y. Acad. Sci. (2004) 1012:115–128.[CrossRef][Web of Science][Medline]
Dolinska M, Zablocka B, Sonnewald U, Albrecht J. Glutamine uptake and expression of mRNA's of glutamine transporting proteins in mouse cerebellar and cerebral cortical astrocytes and neurons. Neurochem. Int. (2004) 44:75–81.[CrossRef][Web of Science][Medline]
Gavin CE, Gunter KK, Gunter TE. Manganese and calcium efflux kinetics in brain mitochondria. Relevance to manganese toxicity. Biochem. J. (1990) 266:329–334.[Web of Science][Medline]
Green DR, Reed JC. Mitochondria and apoptosis. Science (1998) 281:1309–1312.
Gunter TE, Gavin CE, Aschner M, Gunter KK. Speciation of manganese in cells and mitochondria: A search for the proximal cause of manganese neurotoxicity. Neurotoxicology (2006) 27:765–776.[CrossRef][Web of Science][Medline]
HaMai D, Bondy SC. Oxidative basis of manganese neurotoxicity. Ann. N. Y. Acad. Sci. (2004) 1012:129–141.[CrossRef][Web of Science][Medline]
Heckel T, Broer A, Wiesinger H, Lang F, Broer S. Asymmetry of glutamine transporters in cultured neural cells. Neurochem. Int. (2003) 43:289–298.[CrossRef][Web of Science][Medline]
Kadiiska MB, Gladen BC, Baird DD, Graham LB, Parker CE, Ames BN, Basu S, FitzGerald GA, Lawson JA, Marnett LJ, et al. Biomarkers of oxidative stress study III. Effects of the nonsteroidal anti-inflammatory agents indomethacin and meclofenamic acid on measurements of oxidative products of lipids in CCl4 poisoning. Free Radic. Biol. Med. (2005) 38:711–718.[CrossRef][Web of Science][Medline]
Kimelberg HK, Aschner M. Functions of astrocytes. Astrocytes in Brain Aging and Neurodegeneration—Schipper HM, ed. (1998) Montreal, Canada: Landes Bioscience. 15–39.
Kitazawa M, Anantharam V, Yang Y, Hirata Y, Kanthasamy A, Kanthasamy AG. Activation of protein kinase C by proteolytic cleavage contributes to manganese-induced apoptosis in dopaminergic cells: Protective role of Bcl-2. Biochem. Pharmacol. (2005) 69:133–146.[CrossRef][Web of Science][Medline]
Latchoumycandane C, Anantharam V, Kitazawa M, Yang Y, Kanthasamy A, Anumantha Gand, Kanthasamy A. Protein kinase C is a key downstream mediator of manganese-induced apoptosis in dopaminergic neuronal cells. J. Pharmacol. Exp. Ther. (2005) 313:46–55.
Milatovic D, VanRollins M, Li K, Montine KS, Montine TJ. Suppression of murine cerebral F2-isoprostanes and F4-neuroprostanes from excitotoxicity and innate immune response in vivo by alpha- or gamma-tocopherol, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. (2005) 827:88–93.[CrossRef]
Milatovic D, Zaja-Milatovic S, Montine KS, Horner PJ, Montine TJ. Pharmacologic suppression of neuronal oxidative damage and dendritic degeneration following direct activation of glial innate immunity in mouse cerebrum. J. Neurochem. (2003) 87:1518–1526.[Web of Science][Medline]
Morrow JD, Roberts LJ. Mass spectrometric quantification of F2-isoprostanes in biological fluids and tissues as measure of oxidant stress. Methods Enzymol. (1999) 300:3–12. 2nd.[Web of Science][Medline]
Rao KV, Norenberg MD. Manganese induces the mitochondrial permeability transition in cultured astrocytes. J. Biol. Chem. (2004) 279:32333–32338.
Rao VL, Dogan A, Todd KG, Bowen KK, Kim BT, Rothstein JD, Dempsey RJ. Antisense knockdown of the glial glutamate transporter GLT-1, but not the neuronal glutamate transporter EAAC1, excerbates transient focal cerebral ischemia-induced neuronal damage in rat brain. J. Neurosci. (2001) 21:1876–1883.
Racette BA, McGee-Minnich L, Moerlein SM, Mink JW, Videen TO, Perlumutter JS. Welding-related parkinsonism: Clinical features, treatment, and pathophysiology. Neurology (2001) 56:8–13.
Sonnewald U, Westergaard N, Schousboe A. Glutamate transport and metabolism in astrocytes. Glia (1997) 21:56–63.[CrossRef][Web of Science][Medline]
Suzuki Y, Mouri T, Suzuki Y, Nishiyama K, Fujii N. Study of subacute toxicity of manganese dioxide in monkeys. Tokushima J. Exp. Med. (1975) 22:5–10.[Medline]
Takeda A. Manganese action in brain function. Brain Res. Rev. (2003) 41:79–87.[CrossRef][Medline]
Turrens JF, Boveris A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem. J. (1980) 191:421–427.[Web of Science][Medline]
Uchida K. 4-hyroxy-2-nonenal: A product and mediator of oxidative stress. Prog. Lipid Res. (2003) 42:318–343.[CrossRef][Web of Science][Medline]
Wu Q, Sidoryk M, Mutkus L, Zielinska M, Albrecht J, Aschner M. Acrylamide stimulates glutamine uptake in Fischer 344 rat astrocytes by a mechanism involving upregulation of the amino acid transport system N. Ann. N. Y. Acad. Sci. (2005) 1053:435–443.[CrossRef][Web of Science][Medline]
Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones DP, Wang X. Prevention of apoptosis by Bcl-2 release of cytochrome c from mitochondria blocked. Science (1997) 275:1129–1132.
Yang MS, Yu LC, Gupta RC. Analysis of changes in energy and redox states in HepG2 hepatoma and C6 glioma cells upon exposure to cadmium. Toxicology (2004) 201:105–113.[CrossRef][Web of Science][Medline]
Yin Z, Milatovic D, Aschner JL, Syversen T, Rocha JBT, Souza DO, Sidoryk MS, Albrecht J, Aschner M. Methylmercury induces oxidative injury, alterations in permeability and glutamine transport in cultured astrocytes. Brain Res. (2007) 1131:1–10.[CrossRef][Web of Science][Medline]
Zhang S, Fu J, Zhou Z. In vitro effect of manganese chloride exposure on reactive oxygen species generation and respiratory chain complexes activities of mitochondria isolated from rat brain. Toxicol. In Vitro (2004) 18:71–77.[CrossRef][Web of Science][Medline]
Zoratti M, Szabo I. The mitochondrial permeability transition. Biochim. Biophys. Acta. (1995) 1241:139–176.[Medline]
Zwingmann C, Leibfritz D, Hazell AS. Energy metabolism in astrocytes and neurons treated with manganese: Relation among cell-specific energy failure, glucose metabolism, and intercellular trafficking using multinuclear NMR-spectroscopic analysis. J. Cereb. Blood Flow Metab. (2003) 23:756–771.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  E.-S. Y. Lee, Z. Yin, D. Milatovic, H. Jiang, and M. Aschner Estrogen and Tamoxifen Protect against Mn-Induced Toxicity in Rat Cortical Primary Cultures of Neurons and Astrocytes Toxicol. Sci., July 1, 2009; 110(1): 156 - 167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Tamm, F. Sabri, and S. Ceccatelli Mitochondrial-Mediated Apoptosis in Neural Stem Cells Exposed to Manganese Toxicol. Sci., February 1, 2008; 101(2): 310 - 320. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||