Singlet Oxygen Adducts of Cholesterol: Photogeneration and Reductive Turnover in Membrane Systems

Identification of signature products provides a powerful means for establishing whether singlet molecular oxygen (1O2) is a reactive intermediate in a photodynamic process. This approach is particularly attractive for biological systems in which direct physical measurement is difficult because of the short lifetime of 1O2. Among the many possible reporter molecules in a target system, cholesterol (Ch) has the advantage of affording a limited number of readily distinguishable oxidation products, among which are the hydroperoxides 3β‐hydroxy‐5α‐cholest‐6‐ene‐5‐hydroperoxide (5α‐OOH), 3p‐hydroxy‐cholest‐4‐ene‐6a‐hydroperoxide (6α‐OOH) and 3β‐hy‐droxycholest‐4‐ene‐6β‐hydroperoxide (6β‐OOH) that derive specifically from 1O2 addition. The purpose of this study was to compare these species in terms of (1) rates of accumulation in photodynamically treated liposomal membranes; (2) susceptibility to iron‐mediated 1 e‐reduction that triggers chain peroxidative damage; (3) susceptibility to selenoperoxidase (phospholipid hydroperoxide glutathione peroxidase [PHGPX])‐mediated 2 e reduction that protects against such damage and (4) relative toxicity to mammalian cells. Our results indicate that 5α‐OOH is photogenerated at a much greater initial rate than 6α‐OOH or 6β‐OOH. Although liposomal 5α‐OOH, 6α‐OOH, and 6β‐OOH exhibit similar first‐order decay kinetics during iron/ascorbate treatment, the former decays much more slowly during GSH/PHGPX treatment, and is more toxic to L1210 cells. These and related findings suggest that 5α‐OOH is potentially the most damaging ChOOH to arise in photodynamically treated cells.