Surprising Inability of Singlet Oxygen‐generated 6‐Hydroperoxycholesterol to Induce Damaging Free Radical Lipid Peroxidation in Cell Membranes^†

Abstract

Singlet oxygen attack on cholesterol (Ch), a prominent monounsaturated lipid of mammalian cell plasma membranes, gives rise to three hydroperoxide (ChOOH) isomers, 5α-OOH, 6α-OOH and 6β-OOH, the latter two in lower yield than 5α-OOH, and 6α-OOH in lowest yield. A third possible positional isomer, 7α-OOH and 7β-OOH, is produced by free radical attack. In the presence of iron and ascorbate (Fe/AH), 5α-OOH or 6β-OOH in phosphatidylcholine/Ch/ChOOH (20:15:1 by mol) liposomes was reduced to its corresponding alcohol, the rate constant being approximately the same for both ChOOHs. Using [ 14 C]Ch as an in situ probe, we found that liposomal 5α-OOH readily set off free radical-mediated (chain) peroxidation reactions when exposed to Fe/AH, whereas 6β-OOH under the same conditions did not. Moreover, liposomal 5α-OOH triggered robust chain peroxidation in [ 14 C]Ch-labeled L1210 cells, leading to cell death, whereas 6β-OOH was essentially inert in this regard. Thus, 5α-OOH and 6β-OOH undergo iron-catalyzed reductive turnover, but only the former can provoke toxic membrane damage. These novel findings have important implications for UVA-induced photodamage in Ch-rich tissues like skin and eye, where 1 O 2 often plays a major role.

Molecular simulations of the effects of phospholipid and cholesterol peroxidation on lipid membrane properties

“…Recently, it has been shown that both 5-and 6-ChOOH are equally susceptible to iron-catalyzed one electron reduction in membranes, but only 5-ChOOH is able to initiate free radical-mediated lipid peroxidation [47]. It has been suggested that an unusual orientation of the 6-ChO· radical intermediate would preclude hydrogen abstraction reactions that propagate oxidative damage.…”

Cholesterol Hydroperoxide Generation, Translocation, and Reductive Turnover in Biological Systems

“…It was subsequently found that 5α-OOH, 6β-OOH, and 7α-OOH in liposomes underwent one-electron decay at the same rate as one another. However, whereas 5α-OOH and 7α-OOH initiated chain peroxidation robustly in the presence of iron and ascorbate, 6β-OOH was unexpectedly very weak in this regard, possibly due to sluggish initiation by 6β-O· [24]. It appears, therefore, that relatively rapid reduction of 6β-OOH by GPx4/GSH (see above) only partially explains this peroxide’s weak cytotoxicity [24].…”

“…However, whereas 5α-OOH and 7α-OOH initiated chain peroxidation robustly in the presence of iron and ascorbate, 6β-OOH was unexpectedly very weak in this regard, possibly due to sluggish initiation by 6β-O· [24]. It appears, therefore, that relatively rapid reduction of 6β-OOH by GPx4/GSH (see above) only partially explains this peroxide’s weak cytotoxicity [24]. More advanced studies showed that a breast tumor cell line overexpressing GPx4 in mitochondria were much more resistant to 7α/7β-OOH-induced peroxidative injury and apoptotic death than wild type controls, thus demonstrating GPx4’s key role in cytoprotection against these free radical-generated peroxides [20].…”

Effects of UV-A LED light irradiation on growth of cultured RAW 264.7 cells

“…Singlet oxygen also oxidized cholesterol of the mammalian plasma membrane (Korytowski, Schmitt, and Girotti 2010). The addition of reducing metal ions, including Fe 2C and Cu C to POO and P resulted in generation of proteinderived radicals (He et al 2008).…”