Clinically relevant levels of hyperoxia lead to cell cycle arrest and long-term changes in DNA repair in the developing lung

Abstract

<b>Background:</b> Preterms often require life-saving oxygen therapy. The effects of low-dose hyperoxia (O₂) remain largely unexplored. We studied the acute and sustained effects of short-term O₂ on cellular injury and repair in the neonatal lung. <b>Methods and results:</b> Primary lung epithelial cells and fibroblasts myo-(MFB) from 5-7 day-old mice were O₂-exposed (FiO₂=0.4, 24h) followed by RNA sequencing. Transcriptional regulation in both cell types was dominated by the downregulation of genes involved in cell cycle regulation, DNA damage and repair, and developmentally relevant pathways. In MFB, silencing of the top hit <i>Mcm2</i>,&nbsp;critical&nbsp;in DNA replication, resulted in cell cycle arrest and the downregulation of <i>Hif1a</i> and <i>Wnt5a</i>, <i>Acta2</i>, <i>Spp1</i>, and <i>Pdgfra</i>. These genes play important roles in cellular differentiation and migration during lung development, confirmed by altered MFB function. Moreover, a significant downregulation of the DNA damage response genes <i>P21</i>, <i>P53</i>, and <i>Chek1</i> was observed. <i>Apex1</i> expression, key in DNA repair, was abrogated in silenced MFB treated with O₂. Our findings were recapitulated in an independent whole lung single-cell dataset in O_2-exposed mouse pups with downregulation of genes involved in cell cycle regulation and DNA repair. Last, methylation analysis demonstrated regulation of the bases hmC, caC, and 8-OxoG by O₂-exposure in neonatal mice that persists into adulthood. <b>Conclusions:</b> Short-term exposure to low-dose O₂ results in cell cycle arrest, abrogation of developmental signaling, and altered DNA damage and repair in vitro and in vivo persisting long-term, which strongly indicates increased vulnerability to injury hits in later life.