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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 microM FeCl3:100 microM dopamine); (ii) specific uptake of the Fe-dopamine complex into RCHT cells via NET (79+/-2 pmol 59Fe/mg/min; P<0.05), since the uptake of the 59Fe-dopamine complex by the cells was inhibited by 30 microM 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 microM Fe-dopamine complex in the presence of 100 microM dicoumarol, an inhibitor of DT-diaphorase, induced significant cell death (51+/-5%; P<0.001). However, this cell death was reduced by 75% when the cells were incubated in the presence of 30 microM reboxetine (P<0.01). No significant cell death was observed when the cells were incubated with 100 microM dopamine, 100 microM Fe-Dopamine complex, 100 microM dicoumarol, or 100 microM 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 microM 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 microM Fe-dopamine complex in the presence of 100 microM 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.

The role of dopamine in iron uptake into catecholaminergic neurons, and dopamine oxidation to aminochrome and its one-electron reduction in iron-mediated neurotoxicity, was studied in RCSN-3 cells, which express both tyrosine hydroxylase and monoamine transporters. The mean ± SD uptake of 100 lM 59 FeCl 3 in RCSN-3 cells was 25 ± 4 pmol per min per mg, which increased to 28 ± 8 pmol per min per mg when complexed with dopamine (Fe(III)-dopamine). This uptake was inhibited by 2 lM nomifensine (43% p < 0.05), 100 lM imipramine (62% p < 0.01), 30 lM reboxetine (71% p < 0.01) and 2 mM dopamine (84% p < 0.01). The uptake of 59 Fe-dopamine complex was Na + , Cl -and temperature dependent. No toxic effects in RCSN-3 cells were observed when the cells were incubated with 100 lM FeCl 3 alone or complexed with dopamine. However, 100 lM Fe(III)-dopamine in the presence of 100 lM dicoumarol, an inhibitor of DT-diaphorase, induced toxicity (44% cell death; p < 0.001), which was inhibited by 2 lM nomifensine, 30 lM reboxetine and 2 mM norepinephrine. The neuroprotective action of norepinephrine can be explained by (1) its ability to form complexes with Fe 3+ , (2) the uptake of Fe-norepinephrine complex via the norepinephrine transporter and (3) lack of toxicity of the Fe-norepinephrine complex even when DT-diaphorase is inhibited. These results support the proposed neuroprotective role of DT-diaphorase and norepinephrine. Keywords: dopamine, iron, monoamine transporter, neuroprotection, neurotoxicity, norepinephrine. Parkinson's disease is a neurodegenerative disorder characterized by the progressive loss of neurons that comprise the nigrostriatal dopaminergic system. Although it is generally accepted that free radicals are involved in the neurodegenerative process affecting the nigrostriatal system in patients with Parkinson's disease, the exact mechanism of neurodegeneration in vivo is still unknown (Alam et al. 1997;Facchinetti et al. 1998;Jenner 1998;Selley 1998;Ilic et al. 1999;Berg et al. 2001). One possible mechanism for free radical formation involves iron, as total iron levels in the substantia nigra (SN) of patients with Parkinson's disease are reportedly raised compared with those in age-matched controls (Sofic et al. 1988(Sofic et al. , 1991. Iron is indispensable for life, and it is an essential component of heme and non-heme

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Monoamine oxidase-A (MAO-A) [amiflamine (AMF) and 4-methylthioamphetamine (MTA)] and MAO-B (L-deprenyl) inhibitors were found to be cytotoxic in a concentration-dependent manner for RCHT cells derived from adult rat hypothalamus. The cytotoxic effects were increased when the inhibitors were co-incubated with dicoumarol and especially with 25 micro M AMF+100 micro M dicoumarol (2.5-fold; P <0.001). The treatment of RCHT cells solely with AMF induced a marked decrease in the expression of DT-diaphorase mRNA.