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Attenuation of Cytochrome CYP1B1 Expression by Hyperoxia In Human Bronchial Epithelial CELLS BEAS-2B: Implications for Oxygen-Mediated LUNG Injury

Friday, October 19, 2012
Room R02-R05 (Morial Convention Center)
Daniela Dinu, MD, neonatology, fellow, Chun Chu, Weivu Jiang, Binoy Shivanna, Xanthi Couroucli and Bhagavatula Moorthy, Pediatrics, Baylor College of Medicine, Houston, TX


Background: Supplemental oxygen, required to treat premature infants with pulmonary insufficiency, contributes to the development of bronchopulmonary dysplasia (BPD) in animal models and infants. The mechanisms by which oxygen mediates pulmonary injury are not completely understood. We recently observed that cyp1b1-null mice are less susceptible to hyperoxic lung injury, suggesting a pro-oxidant role for CYP1B1. Hyperoxia inhibits the growth of the cells, and β-naphthoflavone (BNF) was reported to protect cells from hyperoxic induced injury.

Objective: In this study, we tested the following hypotheses: 1. hyperoxia attenuates endogenous and inducible CYP1B1expression in the human lung cell line, BEAS-2B; 2. BNF induces CYP1B1 in BEAS-2B cells, thus modulating hyperoxic injury to the cell; and 3. downregulation of CYP1B1 will protect cells from hyperoxic injury.

Methods BEAS-2B cells treated with DMSO (control) or 10 µM BNF were maintained in room air or exposed to hyperoxia for 24, 48, and 72 h. CYP1B1 promoter activity, mRNA and protein expression were evaluated. Cell proliferation and viability were also assessed. Apoptotic markers were measured using TUNEL assay, and reactive oxigen species were measured using flow cytometry.

Results Untreated BEAS-2B cells expressed endogenous CYP1B1 protein, which was diminished by about 50% by 24 or 48 h of hyperoxia exposure. BNF significantly induced both mRNA and protein expression of CYP1B1 in BEAS2B cells. Hyperoxia attenuated endogenous and BNF induced CYP1B1 protein expression and mRNA expression. Also, hyperoxia attenuated luciferase driven CYP1B1 promoter activity. Nevertheless, BNF failed to restore cell growth that has been hampered by hyperoxia, had minimal improvement in cell viability and cells treated with BNF and exposed to hyperoxia had an increase in reactive oxygen species production as well as an increase in apoptotic markers at 72h. Downregulation of CYP1B1 using siRNA improved cells viability in hyperoxia, but did not decrease apoptotic markers.


Our finding that hyperoxia inhibits CYP1B1 protein, mRNA and promoter expression suggests that the attenuation of CYP1B1 protein expression is due to down regulation by transcriptional or post-transcriptional mechanisms. Since CYP1B1 appears to contribute to lung injury mediated by hyperoxia, understanding the mechanisms of regulation of CYP1B1 by hyperoxia may lead to new strategies to prevent or treat BPD