Cytotoxicity of oxidized low-density lipoprotein in cultured RPE cells is dependent on the formation of 7-ketocholesterol.

PURPOSE

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.