Toxicity of 6-hydroxydopamine: live cell imaging of cytoplasmic redox flux.

Cell Biol Toxicol 2012 Apr 8;28(2):89-101. Epub 2012 Jan 8.

Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, FL 34987, USA.

Oxidative stress contributes to several debilitating neurodegenerative diseases. To facilitate direct monitoring of the cytoplasmic oxidation state in neuronal cells, we have developed roTurbo by including several mutations: F223R, A206K, and six of the mutations for superfolder green fluorescent protein. Thus we have generated an improved redox sensor that is much brighter in cells and oxidizes more readily than roGFP2. Cytoplasmic expression of the sensor demonstrated the temporal pattern of 6-hydroxydopamine (6-OHDA) induced oxidative stress in a neuroblastoma cell line (SH-SY5Y). Two distinct oxidation responses were identified in SH-SY5Y cells but a single response observed in cells lacking monoamine transporters (HEK293). While both cell lines exhibited a rapid transient oxidation in response to 6-OHDA, a second oxidative response coincident with cell death was observed only in SH-SY5Y cells, indicating an intracellular metabolism of 6-OHDA, and or its metabolites are involved. In contrast, exogenously applied hydrogen peroxide induced a cellular oxidative response similar to the first oxidation peak, and cell loss was minimal. Glucose deprivation enhanced the oxidative stress induced by 6-OHDA, confirming the pivotal role played by glucose in maintaining a reduced cytoplasmic environment. While these studies support previous findings that catecholamine auto-oxidation products cause oxidative stress, our findings also support studies indicating 6-OHDA induces lethal oxidative stress responses unrelated to production of hydrogen peroxide. Finally, temporal imaging revealed the sporadic nature of the toxicity induced by 6-OHDA in neuroblastoma cells.

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10565-011-9209-3DOI Listing
April 2012
13 Reads

Publication Analysis

Top Keywords

oxidative stress
20
sh-sy5y cells
8
hydrogen peroxide
8
induced 6-ohda
8
oxidative response
8
oxidative
7
6-ohda
6
cells
6
stress
5
cell
5
transporters hek293
4
hek293 cell
4
lacking monoamine
4
cells lacking
4
catecholamine auto-oxidation
4
monoamine transporters
4
findings catecholamine
4
lines exhibited
4
oxidation response
4
response 6-ohda
4

References

(Supplied by CrossRef)

R Andrew et al.
Neurochem Res 1993

A Bellucci et al.
J Neurochem 2008

VV Belousov et al.
Nat Methods 2006

O Ben-Yoseph et al.
J Neurochem 1996

D Blum et al.
Prog Neurobiol 2001

D Blum et al.
Neurosci Lett 2000

H Brenner-Lavie et al.
Biochem Pharmacol 2009

J Callio et al.
J Biol Chem 2005

MB Cannon et al.
Protein Sci 2006

EM Chalovich et al.
J Biol Chem 2006

J Chen et al.
J Biol Chem 2003

Similar Publications