Original Contribution Relationship between oxidative stress and HIF-1α mRNA during sustained hypoxia in humans Vincent Pialoux a,b , Rémi Mounier c,d , Allison D. Brown a,b , Craig D. Steinback a,b , Jean M. Rawling e , Marc J. Poulin a,f,b,g,h,i, ⁎ a Department of Physiology & Biophysics, University of Calgary, Calgary, AB, Canada b Faculty of Medicine, University of Calgary, Calgary, AB, Canada c Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France d Inserm, U567, Paris, France e Calgary Health Region, Calgary, AB, Canada T2N 4N1 f Department Clinical Neurosciences, University of Calgary, Calgary, AB, Canada g Faculty of Kinesiology, University of Calgary, Calgary, AB, Canada h Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada i Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, AB, Canada abstract article info Article history: Received 4 August 2008 Revised 23 October 2008 Accepted 25 October 2008 Available online 10 November 2008 Keywords: Hypoxia DNA oxidation Protein oxidation HIF-1α expression EPO VEGF The aim of this study was to investigate the relations among reactive oxygen species (ROS), hypoxia inducible factor (HIF-1α) gene expression, HIF-1α target gene erythropoietin (EPO), and vascular endothelium growth factor (VEGF) in humans. Five healthy men (32±7 years, mean±SD) were exposed to 12 h of sustained poikilocapnic hypoxia (P ET O 2 =60 mmHg). DNA oxidation (8-hydroxy-2′-deoxyguanosine, 8-OHdG), advanced oxidation protein products (AOPP), EPO, and VEGF were measured in plasma and HIF-1α mRNA was assessed in leukocytes before and after 1, 2, 4, 6, 8,10, and 12 h of exposure to hypoxia. HIF-1α mRNA amount increased during the first two hours of hypoxic exposure and then returned to baseline levels. The findings reveal an up-regulation of HIF-1α (+ 68%), VEGF (+ 46%), and EPO (+74%). AOPP increased continuously from 4 h (+69%) to 12 h (+216%) of hypoxic exposure while 8-OHdG increased after 6 h (+78%) and remained elevated until 12 h. During the “acute” increase phase of HIF-1α (between 0 and 2 h), 8-OHdG was positively correlated with HIF-1α (r = 0.55). These findings suggest that hypoxia induces oxidative stress via an overgeneration of reactive oxygen species (ROS). Finally, this study in humans corroborates the previous in vitro findings demonstrating that ROS is involved in HIF-1α transcription. © 2008 Elsevier Inc. All rights reserved. Introduction Cells exposed to hypoxia respond by transcriptional changes that promote processes such as erythropoeisis and angiogenesis through the activation of hypoxia inducible factor (HIF-1) [29]. HIF-1 is a heterodimeric transcription factor composed of HIF-1α and HIF-1β subunits. In the presence of oxygen, HIF-1α is degraded through the ubiquitin/proteasome pathway mediated by the tumor suppressor protein von Hippel-Lindau (VHL). This is triggered through post- translational hydroxylation via oxygen-dependent prolyl hydroxy- lases (PHDs) within the oxygen-dependent degradation domain [20]. In hypoxia, hydroxylation of HIF-1α is blocked, inducing the stabilization and subsequent transactivation of HIF-1. Concomitantly, hypoxic cells increase paradoxically their mito- chondrial production of reactive oxygen species (ROS) leading to oxidative stress. The primary source of free radical generation in cells during hypoxia has been reported to be due to a decrease in mitochondria redox potential causing a ROS production from the electron transport chain (ETC) mainly at the level of cytochrome III [11]. In addition, oxygen radical release might be enhanced during hypoxic conditions by the activation of xanthine oxidase [33], NAPDH oxidase [15], and phospholipase A 2 [24]. Excess production of these free radicals and ROS in vivo has a variety of damaging effects which include membrane lipid peroxidation, protein oxidation, and DNA degradation [12,21,39]. Free Radical Biology & Medicine 46 (2009) 321–326 Abbreviations: 8-OHdG, 8-hydroxy-2′-deoxyguanosine; AMS, acute mountain sickness; AOPP, advanced oxidation protein products; CO 2 , carbon dioxide; DNA, deoxyribonucleic acid; ECG, electrocardiogram; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; EPO, erythropoietin; ETC, electron transport chain; FeSO 4 , ferrous sulfate; FRAP, ferric reducing antioxidant power; H 2 O 2 , hydrogen peroxide; HIF-1, hypoxia inducible factor; NAPDH, nicotinamide adenine dinucleotide phosphate oxidase; O 2 , dioxygen; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; P ET O 2 , end-tidal oxygen pressure; PHDs, prolyl hydroxylases; P i O 2 , inspired oxygen pressure; RNA, ribonucleic acid; ROS, reactive oxygen species; VEGF, vascular endothelium growth factor; VHL, von Hippel-Lindau. ⁎ Corresponding author. Department of Physiology & Biophysics, University of Calgary, HMRB-212, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. Fax: +1 403 210 8420. E-mail address: poulin@ucalgary.ca (M.J. Poulin). 0891-5849/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2008.10.047 Contents lists available at ScienceDirect Free Radical Biology & Medicine journal homepage: www.elsevier.com/locate/freeradbiomed