Our long term aim is to investigate the mechanisms of ozone toxicity and to develop biomarkers for the detection of ozone exposure. Ozone is emerging as the most important air pollutant in the United States. In contrast to other components of urban air, levels have increased rather than decreased during the past decade. Ozone stands out as the only air pollutant where significant changes in animals and humans can be demonstrated at the air quality standard. In order to develop biomarkers of ozone-exposure we will quantify perturbations of the redox-status of bronchoalveolar lining fluid (retrieved by lavage), alveolar macrophages, and blood plasma from resting and exercising animals exposed to various levels of ozone (between 0 and 3.0 ppm). Alveolar macrophages exposed to ozone in vitro will also be used. Specific and sensitive methods will be employed to quantitate depletion and redox ratios of selected endogenous antioxidants, and oxidative damage to lipids, proteins, and nucleic acids. Peroxidative damage to lipids will be determined by measuring lipid hydroperoxides by HPLC with chemiluminescence detection, and F2-isoprostanes by negative ion chemical ionization gas chromatography/mass spectrometry. To investigate the protective effects of endogenous antioxidants against ozone toxicity, experimental animals will be depleted of vitamin E, selenium, or glutathione, and exposed to ozone. The above endpoints of oxidative perturbations as well as deletions of DNA fragments as described below will be measured. The genetic effects of ozone exposure will be determined with an assay selecting for intrachromosomal recombination causing deletion (DEL) events. DEL recombination in Saccharomyces cerevisiae is readily inducible by mutagens as well as carcinogens which are not detectable with the Ames assays. Agents causing oxidative stress to the cells induce DEL recombination in yeast. A system selecting for deletions in human cells will be constructed, and it will be examined whether ozone enhances the frequency of deletions in this system. Finally, we will test whether deletions are inducible by ozone in vivo in mammals. A recombination system in the mouse involving a gene duplication at the pink-eyed dilute locus will be used. Experiment are currently under way to determine whether X-rays induce the frequency of deletions in the mouse. Experiments with ozone will be performed. The polymerase chain reaction (PCR) technique will be us to quantify the frequency of deletions in isolated cells. The same strategy to quantify the frequency of deletions between duplicated regions in DNA of cells obtained from human subjects after exposure to ozone may be used to develop a sensitive biomarker of ozone exposure in humans.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
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Harvard University
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