Background Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD). NrF2-mediated induction of NADPH Quinone oxydoreductase enzyme (NQO1) is known to protect the tissues from hyperoxic injury. Sulforaphane is a potent inducer of NrF2 and NQO1, and has been shown to improve cell survival under hyperoxic conditions. Objective To test the hypothesis that human pulmonary cells carrying specific SNPs on the human Nrf2 and NQO1 genes will display decreased inducibility by sulforaphane. Design/Methods: H441 cells grown in room air were transfected with plasmids containing the wild type or mutated Nrf2 promoters [-617 C>A, -651 (G>A), and -653 (A>G)] and treated with sulforaphane or DMSO. In another experiment, H441 cells grown in room air were transfected with plasmids containing the wild type or mutated NQO1 promoters [-1221(A>C)] and treated with various concentrations of sulforaphane. Transcriptional activation of Nrf2 and NQO1 were studied using the dual luciferase assay after both experiments. Results: Compared to wild type, all the 3 different SNPs on the NrF2 promoters showed significantly reduced (p ≤ 0.05) basal promoter activity. The promoter activity was also significantly low (p ≤ 0.05) in all 3 SNPs on treatment with 1µM sulforaphane (Fig 1). Compared to wild type NQO1 promoter, the -1221(A>C) showed significantly reduced promoter activity at basal condition and all different concentrations (p ≤ 0.05) of sulforaphane (Fig 2). Conclusions: Our experiments support the hypothesis that human pulmonary cells carrying specific SNPs on the human Nrf2 and NQO1 genes will display decreased inducibility by sulforaphane than those carrying the wild type genes. Further studies on the functional effects of SNPs on Nrf2 gene may help to develop innovative strategies that can lead to the prevention and/or treatment of BPD.
Effect of SNP on inducibility of NrF2 promoter activity by sulforaphane compared to DMSO controls as measured by dual luciferase assay on H441 cells. WT represents wild type and 651 G->A, 617 C->A and 653 A->G are 3 different SNPs that were studied. Values are means ± SE. Significant differences are indicated by *, p ≤ 0.05.
Effect of SNP on inducibility of NrF2 promoter activity by sulforaphane compared to DMSO controls as measured by dual luciferase assay on H441 cells. WT represents wild type and 651 G->A, 617 C->A and 653 A->G are 3 different SNPs that were studied. Values are means ± SE. Significant differences are indicated by *, p ≤ 0.05.
Promoter activity of wild type NQO1 (pNQO) and the mutant NQO1 (-1221(A>C)) measured by dual luciferase assay at different concentrations of sulforaphane. Values are means ± SEM. Significant differences are indicated by *, p ≤ 0.05.
Promoter activity of wild type NQO1 (pNQO) and the mutant NQO1 (-1221(A>C)) measured by dual luciferase assay at different concentrations of sulforaphane. Values are means ± SEM. Significant differences are indicated by *, p ≤ 0.05.
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