Oxidative Stress and Mitochondrial-Mediated Apoptosis in Dopaminergic Cells Exposed to Methylcyclopentadienyl Manganese Tricarbonyl

• DOI: 10.1124/jpet.302.1.26
• Publication Date: 2003-01-10T18:42:35Z
• Authors: Masashi Kitazawa; Jarrad R. Wagner; Michael L. Kirby; Vellareddy Ananth...; Anumantha G. Kant...
• Keywords: 0301 basic medicine; Neurotransmitter Agents; L-Lactate Dehydrogenase; Caspase 3; Cell Survival; Dopamine; Blotting, Western; Apoptosis; DNA Fragmentation; PC12 Cells; Membrane Potentials; Mitochondria; Rats; 3. Good health; Enzyme Activation; Oxidative Stress; 03 medical and health sciences; Proto-Oncogene Proteins c-bcl-2; Caspases; Organometallic Compounds; Animals; Gasoline; In Situ Hybridization

Methylcyclopentadienyl manganese tricarbonyl (MMT), an organic manganese-containing gasoline additive, was investigated to determine whether MMT potentially causes dopaminergic neurotoxic effects. MMT is acutely cytotoxic and dopamine-producing cells (PC-12) seemed to be more susceptible to cytotoxic effects than nondopaminergic cells (striatal gamma-aminobutyric acidergic and cerebellar granule cells). MMT also potently depleted dopamine apparently by cytoplasmic vesicular release to the cytosol, a neurochemical change resembling other dopaminergic neurotoxicants. Generation of reactive oxygen species (ROS), an early effect in toxicant-induced apoptosis, occurred within 15 min of MMT exposure. MMT caused a loss of mitochondrial transmembrane potential (DeltaPsim), a likely source of ROS generation. The ROS signal further activated caspase-3, an important effector caspase, which could be inhibited by antioxidants (Trolox or N-acetyl cysteine). Predepletion of dopamine by using alpha-methyl-p-tyrosine (tyrosine hydroxylase inhibitor) treatment partially prevented caspase-3 activation, denoting a significant dopamine and/or dopamine by-product contribution to initiation of apoptosis. Genomic DNA fragmentation, a terminal hallmark of apoptosis, was induced concentration dependently by MMT but completely prevented by pretreatment with Trolox, deprenyl (monoamine oxidase-B inhibitor), and alpha-methyl-p-tyrosine. A final set of critical experiments was performed to verify the pharmacological studies using a stable Bcl-2-overexpressing PC-12 cell line. Bcl-2-overexpressing cells were significantly refractory to MMT-induced ROS generation, caspase-3 activation, and loss of DeltaPsim and were completely resistant to MMT-induced DNA fragmentation. Taken together, the results presented herein demonstrate that oxidative stress plays an important role in mitochondrial-mediated apoptotic cell death in cultured dopamine-producing cells after exposure to MMT.