Role of Human NADPH Quinone Oxidoreductase (NQO1) in Oxygen-Mediated Cellular Injury and Oxidative DNA Damage in Human Pulmonary Cells.

Rebecca Burke,Guo-Dong Zhou,Nagireddy Putluri,Krithika Lingappan,Bhagavatula Moorthy,Xanthi Couroucli,Vasanta Putluri,Chun Chu,Rachel E Stading,Laura Bravo

doi:10.1155/2021/5544600

Abstract

Supplemental oxygen administration is frequently used in premature infants and adults with pulmonary insufficiency. NADPH quinone oxidoreductase (NQO1) protects cells from oxidative injury by decreasing reactive oxygen species (ROS). In this investigation, we tested the hypothesis that overexpression of NQO1 in BEAS-2B cells will mitigate cell injury and oxidative DNA damage caused by hyperoxia and that A-1221C single nucleotide polymorphism (SNP) in the NQO1 promoter would display altered susceptibility to hyperoxia-mediated toxicity. Using stable transfected BEAS-2B cells, we demonstrated that hyperoxia decreased cell viability in control cells (Ctr), but this effect was differentially mitigated in cells overexpressing NQO1 under the regulation of the CMV viral promoter, the wild-type NQO1 promoter (NQO1-NQO1), or the NQO1 promoter carrying the SNP. Interestingly, hyperoxia decreased the formation of bulky oxidative DNA adducts or 8-hydroxy-2′-deoxyguanosine (8-OHdG) in Ctr cells. qPCR studies showed that mRNA levels of CYP1A1 and NQO1 were inversely related to DNA adduct formation, suggesting the protective role of these enzymes against oxidative DNA injury. In SiRNA experiments entailing the NQO1-NQO1 promoter, hyperoxia caused decreased cell viability, and this effect was potentiated in cells treated with CYP1A1 siRNA. We also found that hyperoxia caused a marked induction of DNA repair genes DDB2 and XPC in Ctr cells, supporting the idea that hyperoxia in part caused attenuation of bulky oxidative DNA lesions by enhancing nucleotide excision repair (NER) pathways. In summary, our data support a protective role for human NQO1 against oxygen-mediated toxicity and oxidative DNA lesions in human pulmonary cells, and protection against toxicity was partially lost in SNP cells. Moreover, we also demonstrate a novel protective role for CYP1A1 in the attenuation of oxidative cells and DNA injury. Future studies on the mechanisms of attenuation of oxidative injury by NQO1 should help in developing novel approaches for the prevention/treatment of ARDS in humans.

Highlights

Supplemental oxygen is an integral part of medical management of pediatric and adult patients with pulmonary insufficiency [1,2,3]
We tested the hypothesis that overexpression of NADPH quinone oxidoreductase 1 (NQO1) in BEAS-2B cells will mitigate cell injury and oxidative DNA damage caused by hyperoxia and that the presence of A1221C single nucleotide polymorphism (SNP) in the NQO1 promoter would display altered susceptibility to hyperoxia-mediated toxicity
Stable cell lines transfected with pcDNA3.1 (Ctr), pCMV-NQO1 (CMV-NQO1), pNQO1NQO1 (NQO1-NQO1), and pSNPNQO1 (SNP) were cultured under room air (RA) or 80% oxygen (O2) for 48 h. qPCR, using OAZ1 as the reference gene, indicated that NQO1 mRNA level was significantly induced by hyperoxia in Ctr cells (Figure 1(a))

Summary

Introduction

Supplemental oxygen is an integral part of medical management of pediatric and adult patients with pulmonary insufficiency [1,2,3]. Mortality due to ARDS is high (35-46%) [8, 9], and recent studies have shown that ARDS is one of the major causes of death due to the COVID-19 infection [10]. Hyperoxia (>95% FiO2) for 72 hours in rodents results in lung inflammation and injury, eventually leading to cell death [4, 12]. ROS generated in hyperoxic conditions lead to profound cell damage through direct DNA damage, lipid peroxidation, protein oxidation, and alteration of transcription factors [4, 12]. Recent studies from our laboratory have shown a protective effect of cytochrome P450 (CYP) 1A enzymes against hyperoxic lung injury in vivo [13,14,15,16,17,18,19,20]

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