Abstract: Slide Presentations |


Anita J. Reddy, MD*; Jacqui Marzec, BS; Wes Gladwell, MS; Steven Kleeberger, PhD
Author and Funding Information

Duke University, Durham, NC

Chest. 2006;130(4_MeetingAbstracts):86S. doi:10.1378/chest.130.4_MeetingAbstracts.86S-a
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PURPOSE: Susceptibility to acute lung injury appears to be multifactorial, including factors such as genetic predisposition. NAD(P)H:quinone oxidoreductase (NQO1)is an enzyme involved in the two-electron reduction of various compounds, which can either increase or decrease the production of reactive oxygen species (ROS). The balance of ROS and antioxidants present in the lung is critical to protection from lung injury in the setting of hyperoxia. We demonstrate that altering NQO1 expression in the setting of hyperoxia enhances pulmonary epithelial cell injury.

METHODS: Bronchial epithelial cells (Beas2B) were transfected with small interfering RNA (siRNA) targetted against NQO1, as well as negative control siRNA. Cells were then exposed to air or hyperoxia for 8 or 12 hours to determine the response to oxidative stress. Cells were then processed for analysis for apoptosis and total cell death by flow cytometry using Annexin V-FITC conjugated probes. Protein was extracted and fractionated by SDS-PAGE to determine the expression of various proteins, including markers of oxidative stress and inflammation. Cells transfected with siRNA were also assayed for ROS production after exposure to hyperoxia. RNA was extracted for microarray analysis as well.

RESULTS: Transfection of NQO1 siRNA causes approximately 70% knockdown of NQO1 expression. Beas2B cells transfected with NQO1 siRNA were protected against apoptosis (8% decrease) and cell death (13% decrease) after exposure to hyperoxia. Analysis of ROS production also indicates that knockdown of NQO1 results in a decrease in ROS production. Microarray analysis of RNA extracted from transfected, exposed cells revealed downregulation of glutathione transferase and key apoptosis proteins.

CONCLUSION: Our studies show that knockdown of NQO1 in vitro results in a decrease in apoptosis, as well as ROS production, in response to hyperoxia. These results suggest that NQO1 plays a key role in promoting cell death, possibly through production of ROS, in hyperoxia-induced lung injury.

CLINICAL IMPLICATIONS: Patients with polymorphisms in NQO1 may be at an increased risk for developing acute lung injury. Treatments could be developed to overcome NQO1 deficiency in hopes of alleviating lung injury.

DISCLOSURE: Anita Reddy, None.

Monday, October 23, 2006

10:30 AM - 12:00 PM




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