Gene expression profiling of human primary astrocytes exposed to manganese chloride indicates selective effects on several functions of the cells

Abstract

Exposure of adult humans to manganese (Mn) has long been known to cause neurotoxicity. Recent evidence also suggests that exposure of children to Mn is associated with developmental neurotoxicity. Astrocytes are critical for the proper functioning of the nervous system, and they play active roles in neurogenesis, synaptogenesis and synaptic neurotransmission. In this report, to help elucidate the molecular events underlying Mn neurotoxicity, we systematically identified the molecular targets of Mn in primary human astrocytes at a genome-wide level, by using microarray gene expression profiling and computational data analysis algorithms. We found that Mn altered the expression of diverse genes ranging from those encoding cytokines and transporters to signal transducers and transcriptional regulators. Particularly, 28 genes encoding proinflammatory chemokines, cytokines and related functions were up-regulated, whereas 15 genes encoding functions involved in DNA replication and repair and cell cycle checkpoint control were down-regulated. Consistent with the increased expression of proinflammatory factors, analysis of common regulators revealed that 16 targets known to be positively affected by the interferon-γ signaling pathway were up-regulated by Mn²⁺. In addition, 68 genes were found to be similarly up- or down-regulated by both Mn²⁺ and hypoxia. These results from genomic analysis are further supported by data from real-time RT-PCR, Western blotting, flow cytometric and toxicological analyses. Together, these analyses show that Mn²⁺ selectively affects cell cycle progression, the expression of hypoxia-responsive genes, and the expression of proinflammatory factors in primary human astrocytes. These results provide important insights into the molecular mechanisms underlying Mn neurotoxicity.

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.