Abstract 1693: Assessment of Nrf2 luciferase reporter assay and heme oxygenase expression as models of oxidative stress in in vitro cultures of lung cells exposed to cigarette smoke condensate

Abstract

Abstract The Nrf2 signaling pathway is an important regulator associated with oxidative stress, xenobiotic metabolism, DNA damage and anti-inflammatory response. A key responsive gene in the Nrf2 regulatory pathway is heme oxygense 1 (HO-1), a cytoprotective enzyme that serves in an antioxidant capacity. An imbalance in antioxidant capacity observed in animal models or individuals with specific Nrf2 and HO-1 polymorphisms has been associated with susceptibility to lung diseases. The objective of this study was to characterize the response of Nrf 2 promoter regulation and HO-1 expression following exposure to cigarette smoke in lung models for oxidative stress. Cigarette smoke condensate (CSC) was prepared from Kentucky 3R4F (K3R4F) reference cigarettes. Nrf2 promoter regulation and HO-1 expression were evaluated in lung cells exposed to K3R4F CSC (0 − 90μg total particulate matter/ml media). Nrf2 promoter regulation was evaluated in transiently-transfected Nrf2 luciferase reporter cells exposed to K3R4F CSC for 0.5, 1, 2, 6 or 24 hours followed by quantification of luciferase activity. HO-1 expression was assessed following exposure to the K3R4F CSC in normal human bronchial epithelial (NHBE) cells for 3, 6 or 24 hours using real-time quantitative RT/PCR and enzyme linked immunoassay (ELISA). Nrf2 reporter cells exhibited dose-dependent increases in promoter activation following CSC exposure. The maximum response was observed between 6 and 24 hours of exposure with 15-, 25-, and 40-fold increases across the exposure range. The specificity of the promoter response was confirmed with co- exposure of the inhibitor all-trans retinoic acid with CSC. The CSC-induced Nrf2 activation was reduced towards control levels in the presence of the inhibitor. CSC also induced dose- and time-dependent increases on HO-1 mRNA and protein levels in NHBE cells. The mRNA changes ranged from 10- to 70-fold with maximal responses observed between 3 and 6 hours. The dynamic range of HO-1 protein response was observed and reached a level of 11-fold increase by 60 μg/ml. Collectively, these responses are consistent with our previously reported study which identified oxidative stress responsive genes in NHBE cells exposed to repetitive applications of CSC. The data suggest that changes in the Nrf2 regulatory pathway may serve as an effective indicator of oxidative stress and the impact of tobacco products in in vitro lung cell culture models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1693.