Effects of 6-hydroxydopamine on mitochondrial function and glutathione status in SH-SY5Y human neuroblastoma cells.
SH-SY5Y human neuroblastoma cells were incubated with 6-hydroxydopamine (6-OHDA) for 4 and 24 h to examine the mechanism of cell death and to determine the time-dependent effects of 6-OHDA on cellular glutathione status. After 4 h, 6-OHDA significantly depleted cellular ATP and GSH concentrations with only slight increases in cell death. GSH:GSSG ratios and mitochondrial membrane potential (Deltapsim) were significantly decreased during 4 h incubations with 6-OHDA. High concentrations of 6-OHDA (100 microM) induced oxidative stress and mitochondrial dysfunction in SH-SY5Y cells within 4 h leading to cell death. In 24 h incubations, 25 and 50 microM 6-OHDA significantly decreased ATP concentrations; however, significant increases in cell death were only observed with 50 microM 6-OHDA. 6-OHDA induced a concentration-dependent increase in GSH and total glutathione concentrations after 24 h. After exposure to 50 microM 6-OHDA, GSH concentrations were increased up to 12-fold after 24 h with no change in the GSH:GSSG ratio. Gene analysis suggests that the increase in GSH concentration was due to increased expression of the GSH synthesis genes glutamate cysteine ligase modifier and catalytic subunits. Our results suggest that 6-OHDA induces oxidative stress in SH-SY5Y cells resulting in an adaptive increase in cellular GSH concentrations.
Tirmenstein
Mark A
MA
Department of Safety Assessment, GlaxoSmithKline, 709 Swedeland Road, UE0360, King of Prussia, PA 19406-0939, USA. mark.2.tirmenstein@gsk.com
Hu
Catherine X
CX
Scicchitano
Marshall S
MS
Narayanan
Padma K
PK
McFarland
David C
DC
Thomas
Heath C
HC
Schwartz
Lester W
LW
D000255:Adenosine Triphosphate / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D018076:DNA, Complementary / Q000096:biosynthesis / Q000302:isolation & purification; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism*; D016627:Oxidopamine / Q000494:pharmacology*; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013565:Sympatholytics / Q000494:pharmacology*; D013997:Time Factors
D018076:DNA, Complementary; D013565:Sympatholytics; D012313:RNA; D016627:Oxidopamine; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D005978:Glutathione