Background Oxygen supplementation is often required to treat pulmonary insufficiency in newborn infants. Acute lung injury caused by hyperoxic therapy contributes to the development of bronchopulmonary dysplasia (BPD). The molecular mechanisms of oxygen-mediated lung injury are not well understood, but reactive oxygen species likely play a role. Hyperoxia induces cytochrome P450 1A1/1A2 (CYP1A1/CYP1A2) in rodent lung and liver and contributes to attenuation of hyperoxic lung injury. Whether this occurs in human tissue is not known. We hypothesized that hyperoxia modulates CYP1A1 expression in human lung.
Methods Studies were performed in the human lung cell lines H441 (ATCC# HTB-174) and BEAS-2B (ATCC# CRL-9609). Each cell line was exposed to hyperoxia (95% oxygen, 5% carbon dioxide) for 24, 48, and 72 hours. Control cells were exposed to room air (5% carbon dioxide). Cells were harvested and CYP1A1 activity was measured using the ethoxyresorufin-O-deethylase (EROD) assay. To determine if modulation of CYP1A1 expression is preceded by activation of the CYP1A1 promoter, we transfected H441 cells with the plasmid pGL3-1A1 containing a 1.6 kb human CYP1A1 promoter and the luciferase reporter gene, followed by exposure of the cells to hyperoxia for selected time points and determination of luciferase activities.
Results Hyperoxia for 24 hours did not significantly alter EROD (CYP1A1) activities compared to room air controls. However, hyperoxia exposure for 48 hours induced CYP1A1 activity 2-3 fold over control in the BEAS-2B and H441 cells. By 72 hours, the EROD activity was diminished to room air values. Transient transfection experiments with human CYP1A1 promoter revealed significant induction of reporter gene expression by hyperoxia, with maximum induction being observed at 8 hours. The induction declined after 24 hours.
Conclusions Hyperoxia induces CYP1A1 in the human lung epithelial cell lines tested, as measured by EROD activity. Results of our transfection experiments support the hypothesis that hyperoxia induces CYP1A1 in these cells by transcriptional activation of the CYP1A1 gene. Future studies will focus on determining the oxygen responsive elements in the human CYP1A1 gene promoter. Understanding the mechanisms of regulation of CYP1A1 gene expression by hyperoxia could lead to the development of strategies to minimize lung injury in infants undergoing supplemental oxygen therapy.
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