DETECTION OF OZONE-INDUCED DNA SINGLE STRAND BREAKS IN MURINE BRONCHOALVEOLAR LAVAGE CELLS ACUTELY EXPOSED IN VIVO

Abstract

Single-strand breaks (SSBs) in DNA have been used a biomarker of oxidative damage. The comet assay, also known as single-cell gel electrophoresis, was used to investigate the ability of ozone (O3) to induce DNA SSBs in murine bronchoalveolar lavage (BAL) cells. The comet assay is more sensitive than other techniques currently utilized for detecting SSBs and requires fewer cells. In the present study, 3 mice were exposed for 3 h to 0.25 ppm of O3, and 3 to 0.5 ppm of O3 for 3 h. Two air-exposed mice served as negative controls. All mice were euthanized 3 h after exposure, at which time BAL cells were recovered from the lungs and stained with ethidium bromide. BAL cells recovered from an air-exposed mouse were exposed to various concentrations of H2O2 in vitro for 1h at 4 C. Excluding cells from the H2O2 group (n = 25), 50 randomly selected BAL cells were graded by comet tail length into 1 of 4 categories: no damage (0 mm), low damage (1-10 mm), medium damage (11-30 mm), and high damage (31 + mm). The nonparametric Wilcoxon rank-sum test was used for statistical analysis, and p values lower than.05 were considered significant. The H2O2 and the 0.25 and 0.5 ppm O3 groups showed statistically significant increases in DNA SSBs as compared to air-exposed controls. The results of this study indicate that (1) O3 induces DNA strand breaks in murine BAL cells at 0.25 and 0.5 ppm, as evidenced by statistically significant increases in the length of comet tails for O3-exposed groups, and (2) the comet assay can be used to assess O3-induced SSBs for in vivo exposures. Therefore, it has the potential as a biomarker for in vivo oxidant exposures.