Ozone exposure in vivo and formation of biologically active oxysterols in the lung.

Ozone toxicity in the lung is thought to be mediated by products derived from the reaction of ozone with components of the lung epithelial lining fluid. Cholesterol is an abundant component of this epithelial lining fluid, and it is susceptible to ozonolysis, yielding several stable products including 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and 5beta,6beta-epoxycholesterol. Both 5beta,6beta-epoxycholesterol and its metabolite, cholestan-6-oxo-3,5-diol, have been shown to cause cytotoxicity in vitro, suggesting that they may be potential mediators of ozone toxicity in vivo. An ozone-sensitive mouse strain, C57BL/6J, was exposed to varying concentrations of ozone (0.5-3.0 ppm), and subsequently the levels of these cholesterol ozonolysis products were quantitated by electrospray ionization mass spectrometry in bronchoalveolar lavage fluid, lavaged cells, and lung homogenate. An ozone dose-dependent formation of these biologically active oxysterols was observed in vivo, supporting a role for these compounds in ozone toxicity. Since the 5beta,6beta-epoxycholesterol metabolite, cholestan-6-oxo-3,5-diol, was isobaric with other cholesterol ozonolysis products, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol condensation product, 3beta-hydroxy-5beta-hydroxy-B-norcholestan-6beta-carboxaldehyde, detailed mass spectral analysis using electron impact ionization was utilized to differentiate these isobaric cholesterol ozonolysis products. The specific detection of cholestan-6-oxo-3,5-diol in lung homogenate after ozone exposure established formation of 5beta,6beta-epoxycholesterol within the lung after exposure to 0.5 ppm ozone.

Formation of biologically active oxysterols during ozonolysis of cholesterol present in lung surfactant.

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 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al, 5-hydroperoxy-B-homo-6-oxa-cholestan-3beta,7a-diol, and 5beta,6beta-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 5beta,6beta-epoxycholesterol, which is only a minor product during ozonolysis of cholesterol in solution. A dose-dependent formation of 5beta,6beta-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-3beta,5alpha,6beta-triol, and more abundant levels of an unexpected metabolite, cholestan-6-oxo-3beta,5alpha-diol. Both 5beta,6beta-epoxycholesterol and cholestan-6-oxo-3beta,5alpha-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.

Direct electrospray tandem mass spectrometry of the unstable hydroperoxy bishemiacetal product derived from cholesterol ozonolysis.

Cholesterol is the most abundant neutral lipid in the epithelial lining fluid of the lower airways of the lung also known as pulmonary surfactant and a potential target for reaction with ambient ozone when inspired into the human lung. The isolated double bond of cholesterol has been shown to be susceptible to attack by ozone, but the major reaction product from cholesterol ozonolysis had been remarkably difficult to structurally characterize. Recently, NMR and X-ray crystallography have been used to suggest the formation of a hydroperoxy, hydroxy hemiacetal product, using various derivatives and models of cholesterol to stabilize this chemically reactive product. Electrospray ionization mass spectrometry was used to study the somewhat unstable ozonolysis product of cholesterol which was found to display unique ionization and fragmentation properties when collisionally activated. The electron-deficient carbon atoms of this highly oxygenated product permitted covalent attachment of an acetate anion during negative ion electrospray ionization, leading to the formation of abundant adduct ions at m/z 511. Surprisingly, positive ions were not readily formed. Collision induced dissociation of the adduct anion yielded a major ion at m/z 477, corresponding to the loss of hydrogen peroxide. The most abundant fragment ion following collisional activation was observed at m/z 93, resulting from a complex rearrangement subsequent to the attack of the hydroperoxide anion on the carbon center of the acetate adduct. Based on the interpretation of the tandem mass spectral data, the major cholesterol ozonization product was characterized as a hydroperoxy, hydroxy hemiacetal derivative, which was consistent with the NMR and X-ray crystallographic studies which were carried out on the more stable methyl ether derivative.