Cytotoxicity of oxidized low-density lipoprotein in cultured RPE cells is dependent on the formation of 7-ketocholesterol.
OBJECTIVE
To determine which components present in oxidized LDL are responsible for the cytotoxicity associated with its internalization by culture ARPE19 cells.


METHODS
ARPE19 cells were grown in 24-well and 96-well plates. Cell viability was measured by MTT and/or adenosine triphosphate (ATP) content. LDL was oxidized with Cu(+2) and oxysterol content analyzed by a novel HPLC method.


RESULTS
OxLDL showed increased cytotoxicity with prolonged oxidation. Analysis of the oxLDL showed a predominance of the 7-oxygenated products, 7 alpha-hydroxycholesterol (7 alpha HCh), 7 beta-hydroxycholesterol (7 beta HCh), and 7-ketocholesterol (7kCh). Addition of these oxysterols to the ARPE19 cell in free form indicated that 7kCh is the most cytotoxic of the oxysterols but at physiologically unrealistic concentrations. Partitioning of individual oxysterols into nonoxidized LDL at concentrations similar to those found in the oxLDL also indicated that 7kCh is the most cytotoxic of the oxysterols. Transition metals are tightly bound by LDL and play an important role in the oxidation of LDL, but do not seem to enhance its cytotoxicity directly.


CONCLUSIONS
Prolonged oxidation of LDL increases the levels of 7kCh due to further oxidation of 7 alpha HCh and 7 beta HCh. The formation of 7KCh seems to be responsible for most of the cytotoxicity associated with oxLDL internalization in ARPE19 cells.
Laboratory of Retinal Cell and Molecular Biology, Section on Mechanisms of Retinal Diseases, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. rodriguezi@nei.nih.gov
Ignacio R
IR
Rodriguez
Shahabuddin
S
Alam
Jung Wha
JW
Lee
D000255:Adenosine Triphosphate / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004357:Drug Synergism; D006801:Humans; D006888:Hydroxycholesterols / Q000378:metabolism; D007653:Ketocholesterols / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000633:toxicity*; D010084:Oxidation-Reduction; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D013997:Time Factors; D028561:Transition Elements / Q000633:toxicity
D006888:Hydroxycholesterols; D007653:Ketocholesterols; D008077:Lipoproteins, LDL; D013778:Tetrazolium Salts; D013844:Thiazoles; D028561:Transition Elements; C099252:oxidized low density lipoprotein; C011724:cholest-5-en-3 beta,7 alpha-diol; D000255:Adenosine Triphosphate; C022616:thiazolyl blue; C003001:7-ketocholesterol