Dopamine-dependent iron toxicity in cells derived from rat hypothalamus

We report a new and specific mechanism for iron-mediated neurotoxicity using RCHT cells, which were derived from rat hypothalamus. RCHT cells exhibit immunofluorescent-positive markers for dopamine beta-hydroxylase and the norepinephrine transporter, NET. In the present study, we observed that iron-induced neurotoxicity in RCHT cells was dependent on (i) formation of an Fe-dopamine complex (100 mu M FeCl3:100 mu M dopamine); (ii) specific uptake of the Fe-dopamine complex into RCHT cells via NET (79 +/- 2 pmol Fe-59/mg/min; P < 0.05), since the uptake of the Fe-59-dopamine complex by the cells was inhibited by 30 mu M reboxetine, a specific NET inhibitor (78% inhibition, P < 0.001); and (iii) intracellular oxidation of dopamine present in the Fe-dopamine complex to aminochrome; (iv) inhibition of DT-diaphorase, since incubation of RCHT cells with 100 mu M Fe-dopamine complex in the presence of 100 mu M dicoumarol, an inhibitor of DT-diaphorase, induced significant cell death (51 +/- 5%; P < 0.061). However, this cell death was reduced by 75% when the cells were incubated in the presence of 30 mu M reboxetine (P < 0.01). No significant cell death was observed when the cells were incubated with 100 mu M dopamine, 100 mu M Fe-Dopamine complex, 100 mu M dicoumarol, or 100 mu M FeCl3 (8.3 +/- 2, 9 +/- 4, 8.5 +/- 3, or 9.7 +/- 2% of control, respectively). ESR studies using the spin trapping agent DMPO showed no formation of hydroxyl radicals when the cells were incubated with 100 mu M FeCl3 alone. However, using the same ESR technique, the formation of hydroxyl radicals and a carbon-centered radical was detected when the cells were incubated with 100 mu M Fe-dopamine complex in the presence of 100 mu M dicoumarol. These studies suggest that iron can induce cell toxicity by a mechanism that requires the formation and NET-mediated uptake of an Fe-dopamine complex, ultimately resulting in the intracellular formation of reactive species.