Gene expression profiling of human primary astrocytes exposed to manganese chloride indicates selective effects on several functions of the cells
Introduction
Manganese (Mn) is an essential metal required for many enzymatic reactions such as those catalyzed by arginase, glutamine synthetase and manganese-dependent superoxide dismutase. However, elevated Mn exposures can lead to its accumulation in the brain and causes serious neurotoxicity (Crossgrove and Zheng, 2004, Dobson et al., 2004, Erikson et al., 2004a, Hazell, 2002, Newland, 1999, Pal et al., 1999). In humans, Mn deficiency is rare whereas Mn neurotoxicity or manganism can occur due to occupational and environmental exposures (Crossgrove and Zheng, 2004, Pal et al., 1999). It is estimated that over 3700 tons of Mn are released into the atm osphere every year, particularly from the gasoline additive, methylcyclopentadienyl manganese tricarbonyl (MMT). Signs of manganism resemble those of idiopathic Parkinson's disease, including dystonia, bradykinesia, rigidity, and tremor (Barbeau, 1984, Newland, 1999, Pal et al., 1999). In exposed humans and monkeys, Mn accumulates at the highest levels in the striatum, globus pallidus, and substantia nigra (Erikson et al., 2004a). In monkeys dosed for 3 months with MnO2, Mn concentrations reached 264 μM in the striatum and 334 μM in the globus pallidus (Suzuki et al., 1975). Mn exposure may also cause developmental neurotoxicity in children. A recent cross-sectional study of 201 10-year-old children living in Araihazar, Bangladesh showed that water Mn was significantly and adversely associated with Performance and Full Scale raw intelligence score (Wasserman et al., 2006).
Previous studies have suggested that Mn2+ can induce oxidative stress and affect iron metabolism and cellular energy metabolism (Dobson et al., 2004, Erikson et al., 2004a, Hazell, 2002, Li et al., 2005, Lu et al., 2005). Nonetheless, the understanding of the specific mechanisms underlying Mn neurotoxicity in humans remains far from clear. In mammals, Mn is absorbed and transported to the brain by the mechanisms responsible for the uptake of divalent metal ions, including the DMT-1-mediated and transferrin-mediated pathways (Erikson and Aschner, 2006, Erikson et al., 2004b). In the brain, astrocytes are a “sink” for Mn, with concentrations 10–50-fold higher than in neurons (Erikson and Aschner, 2006, Erikson et al., 2004b). Mn transport to astrocytes is significantly affected by Fe status. Also, evidence suggests that Mn transport involves multiple pathways that may be competitive or synergistic with iron transport (Crossgrove and Yokel, 2005, Erikson and Aschner, 2006, Erikson et al., 2004b).
In this report, we focus on the potential effects of Mn on human astrocytes. Astrocytes play active roles in many neuronal functions: maintaining ion and pH homeostasis, promoting the synthesis and removal of neurotransmitters, providing glucose supply and antioxidant defense, and regulating synaptic activity, synaptogenesis and neurogenesis (Auld and Robitaille, 2003, Magisretti and Ransom, 2002, Newman, 2003). Astrocytes are sensors of the brain environment, to which they immediately react on the genomic (gene expression) and non-genomic levels (Nedergaard and Dirnagl, 2005). Inflammatory activation of astrocytes or astrocyte dysfunctions are believed to be associated with several chronic neurological diseases, including prion, Alzheimer's and Parkinson's Diseases, and HIV-1 associated dementia (HAD) (Burwinkel et al., 2004, Choi et al., 2005, Meda et al., 2001, Wang et al., 2004). If Mn neurotoxicity is linked with inflammation, it is plausible that the initiating events may occur in astrocytes. One purpose of this study is to test the hypothesis that Mn may induce the expression of inflammatory mediators in astrocytes.
A new powerful way to begin identifying molecular targets of pathogenic insults to cells is to use microarray technology to obtain a general gene expression profile of affected versus control cells.
Candidate affected genes and their functions can then be subjected to in-depth analysis to determine the mechanisms of their responses to a given stimulus. Here we applied this approach to identify candidate genes and gene families in human primary astrocytes that are affected by exposure of the cells to Mn2+. Furthermore, we applied biochemical and toxicological methods to confirm the prominent effects of Mn2+ in primary human astrocytes. Our studies suggest that Mn2+ selectively affects cell cycle progression and the expression of hypoxia-responsive genes and proinflammatory factors. These results provide new insights into the mechanisms underlying Mn neurotoxicity.
Section snippets
Primary human astrocytes and treatment
Astrocytes were isolated from second trimester (14–19 weeks of gestational age) human fetal brains obtained from elective abortions in full compliance with NIH guidelines, as previously described by Volsky and colleagues (Canki et al., 2001, Wang et al., 2004). Homogenous preparations of astrocytes were obtained using high-density culture conditions in the absence of growth factors in F12 Dulbecco's modified Eagles Medium (DMEM-F12) (GIBCO-Invitrogen, Carlsbad, CA) containing 10% fetal bovine
Mn2+ alters the transcript levels of a subset of genes with distinctive functional characteristics
Previous studies of Mn-exposed monkeys and rats showed that Mn concentration in the striatum and globus pallidus can reach concentrations higher than 200 μM (Erikson et al., 2004a, Suzuki et al., 1975). In addition, Mn concentration in astrocytes can be much higher than in neurons. Thus, to maximize the likelihood of identifying Mn targets that may be relevant to human exposures, we chose to examine the effect of 200 μM Mn on primary human astrocytes. We performed microarray gene expression
Discussion
In this report, we applied microarray gene expression profiling and computational algorithms to identify molecular targets of the common environmental toxicant Mn2+ in primary human astrocytes. Further, we used real-time RT-PCR, Western blotting, flow cytometric, and toxicological analyses to confirm the key results from microarray expression analysis. Our analyses provide several new insights into the molecular neurotoxic actions of Mn. Such insights may provide a molecular basis for further
Acknowledgement
This work is supported in part by public health grants HL65568 (LZ) and NS31492 (GB and DJV).
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Contributed to this work equally.