Intracellular dopamine oxidation mediates rotenone-induced apoptosis in PC12 cells¹

Aim: To study the role of dopamine (DA) in rotenone-induced neurotoxicity in PC12 cells.
Methods: Cell viability was assessed by detecting the leakage of
lactate dehydrogenase (LDH) into the medium. Apoptosis rate was measured by
flow cytometry. Caspase-3-like activity was measured by fluorescence assay using
the probe Ac-DEVD-AMC. The level of intracellular hydrogen peroxide and
other peroxides in PC12 cells were quantified by loading cells with 2^’-7^’-
Dichlorodihydrofluorescein diacetate (DCFH-DA) in fluorescence assay. Lactic
acid was measured spectrophotometrically. The DA levels in PC12 cells were
determined by HPLC-ECD.
Results: A 48-h incubation of PC12 cells with rotenone
caused an apoptotic cell death and elevated intracellular reactive oxygen species
(ROS) and lactic acid accumulation. Intracellular DA depletion with reserpine
significantly attenuated rotenone-induced ROS accumulation and apoptotic
cell death. No change was found in rotenone-induced ROS accumulation when
cells were co-treated with deprenyl. Brief treatment with reserpine at the end of
rotenone treatment had no effect on rotenone-induced neurotoxicity. However,
when cells were first incubated with deprenyl, a monoamine oxidase-B inhibitor
for 30 min then co-incubated with rotenone plus deprenyl, a brief treatment with
reserpine enhanced cell injury.
Conclusion: Rotenone-induced apoptosis in PC12
cells was mediated by intracellular dopamine oxidation.