NADPH:quinone oxidoreductase 1 regulates host susceptibility to ozone via isoprostane generation

Abstract

NADPH:quinone oxidoreductase 1 (NQO1) is recognized as a major susceptibility gene for ozone-induced pulmonary toxicity. In the absence of NQO1 as can occur by genetic mutation, the human airway is protected from harmful effects of ozone. We recently reported that NQO1-null mice are protected from airway hyperresponsiveness and pulmonary inflammation following ozone exposure. However, NQO1 regenerates intracellular antioxidants and therefore should protect the individual from oxidative stress. To explain this paradox, we tested whether in the absence of NQO1 ozone exposure results in increased generation of A(2)-isoprostane, a cyclopentenone isoprostane that blunts inflammation. Using GC-MS, we found that NQO1-null mice had greater lung tissue levels of D(2)- and E(2)-isoprostanes, the precursors of J(2)- and A(2)-isoprostanes, both at base line and following ozone exposure compared with congenic wild-type mice. We confirmed in primary cultures of normal human bronchial epithelial cells that A(2)-isoprostane inhibited ozone-induced NF-κB activation and IL-8 regulation. Furthermore, we determined that A(2)-isoprostane covalently modified the active Cys(179) domain in inhibitory κB kinase in the presence of ozone in vitro, thus establishing the biochemical basis for A(2)-isoprostane inhibition of NF-κB. Our results demonstrate that host factors may regulate pulmonary susceptibility to ozone by regulating the generation of A(2)-isoprostanes in the lung. These observations provide the biochemical basis for the epidemiologic observation that NQO1 regulates pulmonary susceptibility to ozone.

FIGURE 1.

Isoprostane quantitation in murine lungs. C57BL/6 wild-type and NQO1-null mice were exposed to filtered air or OZ (1 ppm; 3 h) and then euthanized 24 h later for lung tissue collection. Mouse lungs were snap frozen, stored at −80 °C, and then extracted for E₂- and D₂-isoprostane quantification by GC-NICI-MS. E₂- and D₂-isoprostane quantitation is presented as ng/g of lung tissue (mean ± S.E. (error bars); n = 13–14 animals/group). *, wild-type OZ significantly different from wild-type FA control, p < 0.05; #, NQO1-null FA significantly different from wild-type FA control, p < 0.05.

FIGURE 2.

Time course of ozone-induced IL-8 mRNA expression in NHBE cells in the presence or absence of A₂-isoprostane. NHBE cells cultured at ALI were exposed to FA or OZ over time (0.4 ppm; 0.5–5 h), and total RNA was isolated using TRIzol reagent. IL-8 gene expression was determined using quantitative RT-PCR and normalization to 18 S rRNA, and expression was calculated by the ΔΔCT method (A). Results were expressed as a percentage of the 30-min filtered air control (mean ± S.E. (error bars); n = 10). *, OZ significantly different from corresponding FA control, p < 0.05. NHBE cells cultured at ALI were exposed to filtered air (control) or ozone (0.4 ppm; 2 (B and C) or 5 h (D and E)) in the presence or absence of A₂-isoprostanes (A2) (50 nm or 2.5 μm) for 1 h prior to and then during the exposure. Total RNA was isolated using TRIzol. IL-8 gene expression was determined using quantitative RT-PCR and normalization to 18 S rRNA, and expression was compared with control treatment levels by the ΔΔCT method (mean ± S.E. (error bars); n = 6–7). *, significantly different from control, p < 0.05; #, ozone + A₂-isoprostanes significantly different from ozone alone, p < 0.05.

FIGURE 3.

IL-8 protein concentrations in NHBE cell-conditioned medium following ozone ± A₂-isoP treatment. NHBE cells cultured at ALI were exposed to FA or OZ (0.4 ppm; 5 h) in the presence or absence of A₂-isoP (A2) (2.5 μm) for a 60-min pretreatment and during OZ or FA exposure. Culture medium was collected at the end of OZ exposure for ELISA quantitation of IL-8 (1:5 or 1:10 dilution). Two different NHBE cell donors were evaluated: n = 9 (A) and n = 14 (B); data are summarized as mean ± S.E. (error bars). *, significantly different between OZ and FA, p < 0.001; #, significantly different between OZ and OZ + A₂-isoP, p < 0.05.

FIGURE 4.

Time course of ozone-induced NF-κB activation in NHBE cells in the presence and absence of A₂-isoprostane. NHBE cells cultured at ALI were exposed to FA or OZ (0.4 ppm; 30, 60, and 120 min), and then total cell lysates were collected. Cell lysate proteins (70 μg) were evaluated by Western blot analysis for phosphorylated p65 (p-p65), total p65, and actin as a control for equal loading and transfer (A). The -fold change between the time points was determined by densitometry, and results are presented as a percentage of the corresponding FA control (B). NHBE cells cultured at ALI were exposed to FA or OZ (0.4 ppm; 30 (C and D) or 60 min (E and F)) in the presence or absence of A₂-isoprostanes (A2) (50 nm or 2.5 μm) for 1 h prior to and then during the exposure. At the end of exposure, total cell lysates (60–100 μg) were evaluated by Western blot analysis for phosphorylated p65 (p-p65), total p65, and actin. Results shown are representative of four to six independent experiments. The -fold change between the time points was determined by densitometry, and results are presented as a percentage of the corresponding FA control. Results are summarized graphically (mean ± S.E. (error bars); n = 4–6). *, significantly different from filter air control, p < 0.05; #, ozone + A₂-isoprostanes significantly different from ozone alone, p < 0.05.

FIGURE 5.

Time course of ozone-induced IKK activity in NHBE cells in the presence and absence of A₂-isoprostane. NHBE cells cultured at ALI were exposed to FA or OZ (0.4 ppm; 30, 60, or 120 min), and then total cell lysates (500 μg of protein) were immunoprecipitated with specific antibodies to IKK enzymes. The resultant immunoprecipitates were assayed for IKK enzyme activity using GST-IκBα as a substrate (A). Phosphorylation of IκBα detected by Western analysis was used as a measure of IKK activity (upper panel). The amount of immunoprecipitated IKK enzyme was determined by reprobing the membranes with total anti-IKKβ antibody (lower panel). Western results are representative of at least two independent time course experiments. NHBE cells cultured at ALI were exposed to filtered air (control) or OZ (0.4 ppm; 30 (B and C) or 60 min (D and E)) in the presence or absence of A₂-isoprostanes (A2) (50 nm or 2.5 μm) for 1 h prior to and then during the exposure. At the end of exposure, total cell lysates (500 μg of protein) were immunoprecipitated with specific antibodies to IKK enzyme, and the resultant immunoprecipitates were assayed for IKK enzymatic activity using GST-IκBα as a substrate. Phosphorylation of IκBα detected by Western blot analysis was used as a measure of IKK activity. The amount of immunoprecipitated IKK was determined by reprobing the membranes with total anti-IKKβ antibody. The -fold change between the time points was determined by densitometry, and results are presented as a percentage of the corresponding FA control. Results are summarized graphically from five separate experiments (mean ± S.E. (error bars); n = 5–8). *, significantly different from control, p < 0.05; #, ozone + A₂-isoprostanes significantly different from ozone alone, p < 0.05. Incubation with non-immune polyclonal rabbit IgG failed to immunoprecipitate IKK (A, lane 1, IgG con). CON, control.

FIGURE 6.

Mass spectrometry analysis of IKK Cys¹⁷⁹ sulfoxide modification in the presence of filtered air or ozone and in the presence or absence of A₂-isoprostane. Shown is the LC-MS identification of A₂-isoP-derivatized Cys¹⁷⁹ from synthetic IKKβ peptide Glu¹⁷²–Lys¹⁹⁸. A, experimental and theoretical precursor ion isotope distributions of the Glu¹⁷²–Lys¹⁹⁸ peptide with an A₂-isoP modification ([M + 3H]³⁺, 1115.5789 m/z). Mass accuracy error compared with theoretical was 0.01 ppm. B, MS/MS spectra of A₂-isoP-derivatized (top spectrum) and non-derivatized (bottom spectrum) Glu¹⁷²–Lys¹⁹⁸ peptide ([M + 3H]³⁺ precursor) localizing the A₂-isoP modification to Cys¹⁷⁹. C, extracted ion chromatograms of A₂-isoP-modified Glu¹⁷²–Lys¹⁹⁸ peptide (1115.5789 ± 20 ppm) between ±O₃ and ±A₂-isoP conditions indicated an O₃-catalyzed mechanism of derivatization. O₃ + F₂-isoP did not modify the peptide (+O₃ + F₂). D, theoretical structure of A₂-isoP covalently modified IKKβ Glu¹⁷²–Lys¹⁹⁸ peptide.

FIGURE 7.

Hypothetical mechanism of ozone-induced and NQO1-mediated production of isoprostanes. The presence or absence of NQO1 regulates the redox status of epithelial cells. In the presence of an oxidative stress such as ozone, isoprostanes are generated. The isoprostane generated is dependent on the redox status of the cells with F₂-isoprostane preferred under reducing conditions, whereas A₂-isoprostane is preferred under oxidizing conditions. In the presence of ozone, A₂-isoprostane inhibits NF-κB activation, resulting in blunted IL-8 up-regulation and decreased neutrophilic inflammation. ROS, reactive oxygen species; KC, keratinocyte chemoattractant; PMN, polymorphonuclear neutrophil.