Troglitazone-induced intracellular oxidative stress in rat hepatoma cells: a flow cytometric assessment.
BACKGROUND
Troglitazone (TRO), a thiazolidinedione (TZD) peroxisome proliferator-activated receptor gamma agonist, was recently withdrawn from the market because of rare but serious hepatotoxicity. Previous studies investigating the cytotoxicity of TRO in cultured rat hepatocytes have conjectured about the role of oxidative stress in TRO-induced hepatotoxicity. Therefore, we investigated whether TRO induces oxidative stress and, if so, the portion of the TRO molecule responsible for the induction of oxidative stress.


METHODS
Novikoff rat hepatoma (N1S1) cells were incubated with TRO, troglitazone quinone (TQ), thiazolidinedione-phenoxyacetic acid (TD-PAA) or rosiglitazone (RSG). Membrane peroxidation, intracellular glutathione (GSH) content, and cellular viability were monitored simultaneously by multiparameter flow cytometry.


RESULTS
TRO and TQ increased membrane peroxidation, decreased intracellular GSH, and decreased cell viability in a concentration-dependent manner. In contrast, TD-PAA and RSG neither increased membrane peroxidation nor induced loss of cell viability. In addition, TRO caused a concentration-dependent increase in intracellular superoxide generation accompanied by a collapse in mitochondrial membrane potential.


CONCLUSIONS
Multiparameter flow cytometric evaluation of N1S1 cells indicated that the chromane ring of TRO, rather than the TZD moiety, may be responsible for oxidative stress and suggested that a direct effect on mitochondrial physiology may play a role in TRO-mediated hepatotoxicity.
Narayanan
Padma Kumar
PK
Safety Assessment, GlaxoSmithKline, King of Prussia, Pennsylvania 19406, USA. padma.k.narayanan@gsk.com
Hart
Timothy
T
Elcock
Fiona
F
Zhang
Cindy
C
Hahn
Laura
L
McFarland
David
D
Schwartz
Lester
L
Morgan
D Gwyn
DG
Bugelski
Peter
P
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002839:Chromans / Q000494:pharmacology*; D004396:Coloring Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005343:Fibrinolytic Agents / Q000494:pharmacology; D005434:Flow Cytometry; D005452:Fluoresceins / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D008956:Models, Chemical; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D010617:Phenanthridines / Q000737:chemistry; D011419:Propidium / Q000494:pharmacology; D051381:Rats; D000077154:Rosiglitazone; D013481:Superoxides / Q000378:metabolism; D045162:Thiazolidinediones / Q000494:pharmacology*; D000077288:Troglitazone
D002839:Chromans; D004396:Coloring Agents; D005343:Fibrinolytic Agents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010617:Phenanthridines; D045162:Thiazolidinediones; D000077154:Rosiglitazone; C074109:5-(N-dodecanoyl)aminofluorescein; D013481:Superoxides; D011419:Propidium; C058428:hydroethidine; D005978:Glutathione; D000077288:Troglitazone; D010100:Oxygen