Increased manganese uptake by primary astrocyte cultures with altered iron status is mediated primarily by divalent metal transporter

Neurotoxicity due to excessive brain manganese (Mn) accumulation can occur via occupational exposure to aerosols or dusts that contain extremely high levels (>1-5 mg Mn/m(3)) of Mn, or metabolic aberrations (decreased biliary excretion). Given the putative role of astrocytes in regulating the movement of metals across the blood-brain barrier, we sought to examine the relationship between iron (Fe) status and Mn transport in astrocytes. Furthermore, our study examined the effect of Fe status on astrocytic transferrin receptor (TfR) and divalent metal transporter (DMT-1) levels and their relationship to Mn uptake, as both have been implicated as putative Mn transporters. All experiments were carried out in primary astrocyte cultures derived from neonatal rats when the cells reached full confluency (about three weeks in culture). Astrocytes were incubated for 24h in astrocyte growth medium (AGM) containing 200 microM desferroxamine (ID), 500 microM ferrous sulfate (+Fe), or no compound (CN). After 24h, 5 min (54)Mn uptake was measured and protein was harvested from parallel culture plates for DMT-1 and TfR immunoblot analysis. Both iron deprivation (ID) and iron overload (+Fe) caused significant increases (p<0.05) in (54)Mn uptake in astrocytes. TfR levels were significantly increased (p<0.05) due to ID and decreased in astrocytes exposed to +Fe treatments. As expected, DMT-1 was increased due to Fe deprivation, but surprisingly, DMT-1 levels were also increased due to +Fe treatment, albeit not to the extent noted in ID. The decreased TfR associated with +Fe treatment and the increased DMT-1 levels suggest that DMT-1 is a likely putative transporter of Mn in astrocytes.