Oxidative stress and mitochondrial-mediated apoptosis in dopaminergic cells exposed to methylcyclopentadienyl manganese tricarbonyl.
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.
Kitazawa
Masashi
M
Parkinson Disorders Research Laboratory, Department of Biomedical Sciences, Iowa State University, Ames, IA 50011-1250, USA.
Wagner
Jarrad R
JR
Kirby
Michael L
ML
Anantharam
Vellareddy
V
Kanthasamy
Anumantha G
AG
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004298:Dopamine / Q000378:metabolism / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D005742:Gasoline / Q000633:toxicity*; D017403:In Situ Hybridization; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D018377:Neurotransmitter Agents / Q000378:metabolism; D009942:Organometallic Compounds / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D016716:PC12 Cells; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism
D005742:Gasoline; D018377:Neurotransmitter Agents; D009942:Organometallic Compounds; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; C009907:2-methylcyclopentadienyl manganese tricarbonyl; D007770:L-Lactate Dehydrogenase; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D004298:Dopamine