COMPARATIVE EFFECTS OF LINOLEIC-ACID AND LINOLEIC-ACID HYDROPEROXIDE ON GROWTH AND MORPHOLOGY OF BOVINE RETINAL-PIGMENT EPITHELIAL-CELLS IN-VITRO

Purpose. Outer segments of the photoreceptor rods that are phagocytize d by the retinal pigment epithelial (RPE) cells contain a high proport ion of polyunsaturated fatty acids (PUFA). PUFA are susceptible to lip id peroxidation. We hypothesized that the resulting peroxides could in jure RPE cells leading to retinal degeneration. Accordingly, we compar ed the effects of linoleic acid (LA) and its hydroperoxide (LHP) on th e growth and morphology of RPE cells using laser scanning microscopy a nd transmission microscopy. Methods. We counted the number of RPE cell s after incubation for 24 and 48 hrs with concentrations of LA or LHP of 0.035, 0.175, and 0.35 mM. To observe the actin filaments, cultured RPE cells were stained with rhodamine phalloidin. The cells were pref ixed with 2% glutaraldehyde and postfixed in 1% osmium tetroxide. Spec imens were embedded in Epon 812 after dehydration, and the ultrathin s ections were doubly stained with 2% uranyl acetate and 2% lead acetate for examination by transmission electron microscopy. Results. Exposur e to LA or LHP produced dose-dependent damage to RPE cells with a sign ificantly greater effects of LHP than LA. After incubation for 24 hrs with 0.35 mM LA, the number of vacuoles in RPE cells exceeded that obs erved in control RPE cells by 365 nm laser microscopy. Exposure to 0.3 5 mM LHP for 24 hrs produced a pycnotic nucleus, with diffuse and gran ular autofluorescences observed in and around it. Exposure of RPE cell s to 0.35 mM LA for 24 hrs showed that the LA incorporated into the ly sosomes was digested and released extra-cellularly from lysosomes via exocytotic vesicles. However, such exposure to LHP damaged the RPE cel ls, including the membranes in the pinocytotic vesicles. The packed me mbranes resembled myelin. Conclusions. While the LA incorporated into the lysosomes was released extracellularly, LHP persisted in the RPE c ells, being observed as autofluorescent lipofuscin-like materials. LHP was cytotoxic, and caused damage to the membranes of pinocytotic vesi cles and lysosomes.