Oxidant-induced apoptosis in cultured human retinal pigment epithelial cells.
OBJECTIVE
To determine the mechanism of oxidant-induced cell death in cultured human retinal pigment epithelium (hRPE).


METHODS
Cultured hRPE cells were treated with different concentrations of a chemical oxidant, t-butylhydroperoxide (tBH), for different periods of time. Apoptosis was determined with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and flow cytometry. Mitochondrial membrane potential (mtdelta psi) was measured by rhodamine 123 staining and subsequent flow cytometry. Release of mitochondrial cytochrome c (cyt c) and cleavage of procaspase 3 and caspase substrates were determined by western blot analysis.


RESULTS
t-Butylhydroperoxide caused time- and dose-dependent activation of apoptosis in hRPE, indicated by characteristic morphologic changes; TUNEL-positive labeling; phosphatidylserine (PS) exposure; and procaspase 3, poly(ADP-ribose)polymerase, lamin, and tubulin cleavage. An early decrease of mtdelta psi was observed before caspase activation, together with the release of mitochondrial cyt c.


CONCLUSIONS
Results indicate that tBH can induce apoptosis in hRPE, probably by triggering the mitochondrial permeability transition, which results in swelling and release of mitochondrial intermembrane proteins.
Cai
J
J
Department of Biochemistry, School of Medicine, Emory University, Atlanta, Georgia 30322, USA.
Wu
M
M
Nelson
K C
KC
Sternberg
P
P
Jones
D P
DP
D000368:Aged; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D008564:Membrane Potentials / Q000502:physiology; D018613:Microscopy, Confocal; D008875:Middle Aged; D008928:Mitochondria / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D020112:Rhodamine 123; D013997:Time Factors; D020122:tert-Butylhydroperoxide / Q000494:pharmacology*
D017304:Annexin A5; D003574:Cytochrome c Group; D020112:Rhodamine 123; D020122:tert-Butylhydroperoxide; D020169:Caspases