Formation of Biologically Active Oxysterols during Ozonolysis of Cholesterol Present in Lung Surfactant

Abstract

Exposure of the lung to concentrations of ozone found in ambient air is known to cause toxicity to the epithelial cells of the lung. Because of the chemical reactivity of ozone, it likely reacts with target molecules in pulmonary surfactant, a lipid-rich material that lines the epithelial cells in the airways. Phospholipids containing unsaturated fatty acyl groups and cholesterol would be susceptible to attack by ozone, which may lead to the formation of cytotoxic products. Whereas free radicalderived oxidized cholesterol products have been frequently studied for their cytotoxic effects, ozonized cholesterol products have not been studied, although they could reasonably play a role in the toxicity of ozone. The reaction of ozone with cholesterol yielded a complex series of products including 3␤-hydroxy-5-oxo-5,6-secocholestan-6-al, 5-hydroperoxy-B-homo-6-oxa-cholestan-3␤,7a-diol, and 5␤,6␤-epoxycholesterol. Mass spectrometry and radioactive monitoring were used to identify the major cholesterol-derived product during the reaction of 2 ppm ozone in surfactant as 5␤,6␤-epoxycholesterol, which is only a minor product during ozonolysis of cholesterol in solution. A dose-dependent formation of 5␤,6␤-epoxycholesterol was also seen during direct exposure of intact cultured human bronchial epithelial cells (16-HBE) to ozone. Studies of the metabolism of this epoxide in lung epithelial cells yielded small amounts of the expected metabolite, cholestan-3␤,5␣,6␤-triol, and more abundant levels of an unexpected metabolite, cholestan-6-oxo-3␤,5␣-diol. Both 5␤,6␤-epoxycholesterol and cholestan-6-oxo-3␤,5␣-diol were shown to be cytotoxic to cultured 16-HBE cells. A possible mechanism for cytotoxicity is the ability of these oxysterols to inhibit isoprenoid-based cholesterol biosynthesis in these cells.Human exposure to 0.2 ppm levels of ozone in ambient air has been shown to cause numerous pulmonary effects such as increased airway inflammation and decreased pulmonary function (1, 2). Studies of ozone in animals using up to 3 ppm ozone have been shown to cause increased airway hyperresponsiveness and epithelial cell death. It has been hypothesized that the very high chemical reactivity of ozone limits the distribution of this gas in the pulmonary system, preventing direct exposure to the cellular components of the lung. In part ozone may react with the various components of the epithelial cell lining fluid in the lung, also known as pulmonary surfactant, which includes proteins, lipids, and single electron antioxidant agents such as ascorbic acid (3-5). Because of the very high reactivity of ozone with lipids containing double bonds, considerable emphasis has been placed on the reaction of ozone with lipid compounds in the lungs and the possibility that the adverse effects of ozone are mediated by lipid-ozonized products (6). Evidence in support of this theory has been accumulating with the identification of biologically active phospholipids (7) such as 1-hexadecanoyl-2-(9-oxo-nonanoyl)-glycerophosphocholine, found following oz...

Cholesterol, Reactive Oxygen Species, and the Formation of Biologically Active Mediators

“…ceptible to ozone due to the presence of ozone in the troposphere and therefore present in air inspired into the lung. Recent studies have revealed the formation of two major products when low concentrations (100 -500 ppb) of ozone (such levels can be present in the air of large cities, to which humans are exposed on a constant basis) were exposed to lung surfactant (20). The initial reaction of ozone is the addition across the ⌬ 5 -double bond of Ch, which yields a 1,2,3-trioxolane that undergoes rapid and spontaneous decomposition to yield several additional reactive entities, including carbonyl oxides, carbon-centered radicals, and hydroperoxides, which ultimately lead to the cleavage of the C-5-C-6 double bond (Fig.…”

Analysis of oxysterols by electrospray tandem mass spectrometry

“…It is not known if some of these occur naturally, and there is a need for methods to make comprehensive studies of the nature and levels of oxysterols in tissues and body fluids. This is illustrated by the recent identification of novel oxysterols, formed from cholesterol by the action of ozone, in human tissues [27][28][29][30]. Evidence has been presented for their involvement in the mediation of inflammatory processes associated with the development of atherosclerotic lesions [28], and in the initiation of protein misfolding as occurs in several neurological diseases [29].…”

“…The levels of the 5,6-secosterol and its aldol were surprisingly high in brain of patients with Alzheimer's disease and healthy subjects [combined seco-sterol levels ϳ0.44 pmol/mg (ϳ180 ng/g) and ϳ0.35 pmol/mg (ϳ150 ng/g), respectively]. Biologically active oxysterols are also formed during ozonolysis of cholesterol in lung surfactant [30]. Pulfer and Murphy [30] identified the major cholesterol derived ozonolysis product in lung surfactant as 5␤,6␤-epoxycholesterol (C-3␤-ol-5␤,6␤-epoxide).…”

“…Biologically active oxysterols are also formed during ozonolysis of cholesterol in lung surfactant [30]. Pulfer and Murphy [30] identified the major cholesterol derived ozonolysis product in lung surfactant as 5␤,6␤-epoxycholesterol (C-3␤-ol-5␤,6␤-epoxide). In studies of the metabolism of this oxysterol in lung epithelial cells they found small amounts of the expected metabolite cholestane-3␤, 5␣, 6␤-triol (C-3␤, 5␣, 6␤-triol), and more abundant levels of the unexpected metabolite 3␤, 5␣-dihydroxycholestan-6-one (C-3␤, 5␣-diol-6-one).…”

Oxysterols Inhibit Phosphatidylcholine Synthesis via ERK Docking and Phosphorylation of CTP:Phosphocholine Cytidylyltransferase

“…Oxysterols are oxygenated derivatives of cholesterol and important constituents of oxidized low density lipoproteins that are detected in association with surfactant during acute lung injury (4). Oxysterols are present in human lung and exert potent biological effects by controlling expression of diverse genes via binding to liver X receptors (LXRs), members of the nuclear receptor superfamily (5,6).…”