Photodamage Generates 7‐keto‐ and 7‐hydroxycholesterol in the Rat Retina via a Free Radical‐mediated Mechanism

Abstract

Albino Sprague–Dawley rats are known to undergo photoreceptor degeneration after exposure to constant light, but the molecular mechanism(s) by which the photoreceptors degenerate is not fully understood. We hypothesized that cytotoxic oxysterols are generated in situ in the retina under such conditions and may be involved in the degenerative mechanism. Thus, photodamaged and control rat retinas were analyzed for oxysterols by liquid chromatography mass spectroscopy. Elevated levels of two known cytotoxic oxysterols, 7-ketocholesterol (7KCh) and 7αβ-hydroxycholesterol (7HCh), were found in the photodamaged retinas, at levels six-fold and 50-fold greater, respectively, than those found in non photodamaged controls. Notably, two key intermediates, 5,6α,β-epoxycholesterol (5,6-epoxyCh) and 7αβ-hydroperoxy-cholesterol, were also identified, indicating that the formation of 7KCh and 7HCh is mediated by a free radical mechanism. By immunohistochemistry, 7KCh was localized to the ganglion cell layer, photoreceptor inner segments and retinal pigment epithelium (RPE), which coincides with the localization of ferritin in the retina. Exposure of a mixture of ferritin and low-density lipoprotein to intense white light in vitro produced similar oxysterol species as seen in vivo. We propose that the increased levels of 7KCh and 7HCh, especially in photoreceptor inner segments and RPE, may arise due to ferritin-catalyzed reactions and may be important contributors to the photoreceptor degeneration observed in photodamaged rats.

Cholesterol oxidation in the retina: implications of 7KCh formation in chronic inflammation and age-related macular degeneration

“…In monkeys, the levels of 7KCh are between 0.5 and 1.5 pmol/nmol of cholesterol ( 9 ). In albino rats (not photodamaged), the levels are between 1 and 4 pmol/nmol of cholesterol ( 10 ). The levels of other oxysterols were below our limit of detection or <100 fmol/nmol of cholesterol.…”

“…In both mechanisms, 7OOCh leads to the formation of 7HCh and 7KCh. At this time we are unaware of any scientifi c evidence to support the existence of the singlet oxygen mechanism in the retina; only the free radical seems to occur during light damage ( 10 ). B: The enzymatic mechanisms for cholesterol oxidation in the retina are speculative.…”

Brain Cholesterol Synthesis and Metabolism is Progressively Disturbed in the R6/1 Mouse Model of Huntington’s Disease: A Targeted GC-MS/MS Sterol Analysis

“…Free radical mediated oxidation of cholesterol in the 5,6 position forms 7β-hydroxycholesterol (7β-OHC) and 7-ketocholesterol (7-KC). An elevation of these cholesterol oxidation products (COPs) has been reported in several diseases and pathological models that involve oxidative stress [35][36][37][38].…”

Conversion of 7-Dehydrocholesterol to 7-Ketocholesterol Is Catalyzed by Human Cytochrome P450 7A1 and Occurs by Direct Oxidation without an Epoxide Intermediate

“…The level of 7-ketocholesterol in human plasma is about one-half that of 7␣-hydroxycholesterol, the product of the first committed step of bile acid synthesis (16,(25)(26)(27). 7-Ketocholesterol is one of the major oxysterols in human atherosclerotic plaques and accumulates in photodamaged rat retina (15,28). Levels of 7-ketocholesterol are also elevated in patients with some inherited diseases, e.g.…”

“…Cholesterol has been the presumed precursor of 7-ketocholesterol, but the mechanism of formation of 7-ketocholesterol in the body is unclear. Much of the literature attributes its source to lipid peroxidation and other radical processes acting on cholesterol (16,28,(32)(33)(34)(35). A 7-hydroxycholesterol dehydrogenase was purified from hamster liver microsomes, although the enzyme is apparently not present in rats or (most) humans (36).…”