Ozone Exposure in Vivo and Formation of Biologically Active Oxysterols in the Lung

Abstract

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 3␤-hydroxy-5-oxo-5,6-secocholestan-6-al and 5␤,6␤-epoxycholesterol. Both 5␤,6␤-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 dosedependent formation of these biologically active oxysterols was observed in vivo, supporting a role for these compounds in ozone toxicity. Since the 5␤,6␤-epoxycholesterol metabolite, cholestan-6-oxo-3,5-diol, was isobaric with other cholesterol ozonolysis products, 3␤-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol condensation product, 3␤-hydroxy-5␤-hydroxy-B-norcholestan-6␤-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 5␤,6␤-epoxycholesterol within the lung after exposure to 0.5 ppm ozone.

A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma

“…Recently, methods have been developed for sterol analysis using LC-MS (24)(25)(26). Like GC and GC-MS, these methods are typically optimized for the detection of one or two analytes.…”

Protection against inhaled oxidants through scavenging of oxidized lipids by macrophage receptors MARCO and SR-AI/II

“…Also, β-epoxide is created from cholesterol in cell membranes. Both lipids are detected in vitro after exposing bovine surfactant to ozone (4,5), and β-epoxide and nonanals are detected in vivo in BALs after exposing rodents to ozone (17,30). We are not aware of previous studies measuring β-epoxide and PON-GPC using the 48-hour exposure conditions of the present study.…”

“…Ozone exposure of lung lining fluid cholesterol creates β-epoxide, an oxidized surfactant lipid that is cytotoxic in vitro and may contribute to lung inflammation after ozone inhalation (4,17). Also, ozonolysis of unsaturated surfactant glycerophosphocholines creates PON-GPC (5).…”

“…We are not aware of previous studies measuring β-epoxide and PON-GPC using the 48-hour exposure conditions of the present study. However, exposing C57BL/6 mice to 0.5 ppm ozone for 3 hours creates 2.5 ng β-epoxide per ml BAL, and unexposed lungs from C57BL/6 mice have β-epoxide levels of approximately 95 ng per lung (17). Our data using bolus instillation of 1 μg β-epoxide and PON-GPC provide a proof-of-principle, but future studies would benefit by more detailed measurement of the BAL levels of oxidized lipids during the 48-hour exposure to ozone.…”

Scavenger receptors clear the air

“…In these studies (10), unique ozonation product 1-palmitoyl-2-(9'-oxo-nonanoyl)-glycerophosphocholine (PON-GPC) and a nonspecific autoxidation product 5β,6β-epoxycholesterol, which are produced during ozone exposure (11,12), were directly instilled into wild-type as well as MARCO and SR-AI/II knockout mice. Scavenger receptors were found to affect inflammation, as measured by polymorphonuclear…”