A toxicogenomic profile of Mn in Human Astrocytes

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 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. These results are further supported by data from real-time RT-PCR, Western blotting and flow cytometric analyses. In addition, analysis of common regulators revealed that 16 targets known to be positively affected by the IFN-gamma signaling pathway were up-regulated by Mn, suggesting that the proinflammatory IFN-gamma signaling pathway was likely activated. These results raise the possibility that inflammatory activation of astrocytes and the increased expression of proinflammatory cytokines and chemokines and/or the activation of related signaling pathways might be an important mechanism leading to Mn neurotoxicity. Keywords: treatment comparison Primary human astrocytes (passage 4) were treated with or without 200 uM MnCl2 for 7 days. Total RNA was extracted by using TRIzol reagent (GIBCOBRL Life Technologies). The quality of RNA was high as assessed by measuring absorbance at 260 and 280 nm, by gel electrophoresis, and by the quality of microarray data (see below). We isolated RNA from three independent batches of human astrocytes, which were from different subjects. No identifiable information from these subjects was available, in keeping with the guidelines on human subjects. Three independent batches of astrocytes from three different subjects, not the same subject, were used to ensure that our data and conclusions would not totally rely on one unidentified subject, who may have experienced influential environmental or genetic conditions. The synthesis of cDNAs and biotin-labeled cRNAs were carried out exactly as described in the Affymetrix GeneChip Expression Analysis Technical Manual (2000). The human genome U133 plus 2.0 arrays were purchased from Affymetrix, Inc. Probe hybridization and data collection were carried out by the Columbia University Affymetrix GeneChip processing center. Initial data analysis was performed by using the Affymetrix Microarray suite.