Diffusion of singlet oxygen into human bronchial epithelial cells

The respiratory epithelium undergoes morphological and functional changes following exposure to single oxygen. However, mechanisms by which singlet oxygen causes cellular injury are unclear. The present experiments were designed to investigate the possibility that singlet oxygen, a highly reactive species, diffuses into respiratory epithelial cells. Of the various methods for detection of singlet oxygen, the electron spin resonance (ESR) spectrometric technique was judged to be most compatible and sensitive for use with cell suspensions. ESR spectrometry was used to monitor the singlet oxygen-mediated conversion of 2-(9,10-dimethoxyanthracenyl)-tert-butylhydroxylamine, (I), to 2-(9,10-dimethoxyanthracenyl)-tert-butylnitroxide, (II), and its corresponding endoperoxide, (III), in human bronchial epithelial cells treated with extracellularly generated singlet oxygen. In a second series of experiments, bronchial epithelial cells labeled with (I) were treated with singlet oxygen in the presence of 1,4-diazabicyclo[2.2.2]octane, a singlet oxygen quenching agent. The addition of this quenching agent eliminated the ESR spectrum corresponding with (II) and (III). This result is consistent with the quenching of singlet oxygen by 1.4-diazabicyclo[2.2.2]octane. Collectively, our results indicate that extracellularly generated singlet oxygen diffuses into human bronchial epithelial cells and that this process is a potentially important step in the cytotoxic action of singlet oxygen to the respiratory epithelium.