Mutation Research 729 (2012) 52–60 Contents lists available at SciVerse ScienceDirect Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis jou rnal h omepa g e: www.elsevier.com/locate/molmut Co mm unit y ad d ress: www.elsevier.com/locate/mutres Role of extracellular DNA oxidative modification in radiation induced bystander effects in human endotheliocytes Svetlana V. Kostyuk a , Aleksei V. Ermakov a,∗ , Anna Yu. Alekseeva a , Tatiana D. Smirnova a , Kristina V. Glebova a , Liudmila V. Efremova a , Ancha Baranova a,b , Natalya N. Veiko a a Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russia b School of System Biology, George Mason University, Fairfax, VA 22030, USA a r t i c l e i n f o Article history: Received 10 April 2011 Received in revised form 30 August 2011 Accepted 27 September 2011 Available online 4 October 2011 Keywords: Human umbilical vein endothelial cells (HUVECs) Extracellular DNA Low doses of irradiation Bystander effect 8-Oxo-guanosine a b s t r a c t The development of the bystander effect induced by low doses of irradiation in human umbilical vein endothelial cells (HUVECs) depends on extracellular DNA (ecDNA) signaling pathway. We found that the changes in the levels of ROS and NO production by human endothelial cells are components of the radia- tion induced bystander effect that can be registered at a low dose. We exposed HUVECs to X-ray radiation and studied effects of ecDNA R isolated from the culture media conditioned by the short-term incubation of irradiated cells on intact HUVECs. Effects of ecDNA R produced by irradiated cells on ROS and NO pro- duction in non-irradiated HUVECs are similar to bystander effect. These effects at least partially depend on TLR9 signaling. We compared the production of the nitric oxide and the ROS in human endothelial cells that were (1) irradiated at a low dose; (2) exposed to the ecDNA R extracted from the media conditioned by irradiated cells; and (3) exposed to human DNA oxidized in vitro. We found that the cellular responses to all three stimuli described above are essentially similar. We conclude that irradiation-related oxidation of the ecDNA is an important component of the ecDNA-mediated bystander effect. © 2011 Elsevier B.V. All rights reserved. 1. Introduction In living cells, an ionizing radiation can produce both direct intracellular damage by the deposition of energy into the target molecules and indirect effects induced by radicals formed after water radiolysis. The latter includes the production of reactive oxy- gen species (OH • , O 2 •- , 1 O 2 , NO • , NO 2 • , ONOO - , HO 2 • , H 2 O 2 ) giving rise to oxidative stress. However, the synthesis of these compounds continues for at least 2–5 min after irradiation [1,2]. ROS and RNS cause various DNA lesions, including base modifications, damage to deoxyriboses, generation of the apurinic/apyrimidinic sites, sin- gle and double strand breaks, crosslinks with proteins and other lesions [3–6]. As an example, an irradiation of the endothelial cells at a dose of 0.1 Gy leads to the doubling of the amount of the cells with double strand DNA breaks and apoptotic cells [7]. Low dose exposures to ionizing radiation involve bystander effects (BE), i.e. non-targeted and delayed effects [8–10]. These responses are seen within the population of non-irradiated bystander cells. A number of mechanisms explaining the BE ∗ Corresponding author at: Laboratory of Molecular Biology, Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moskvorechye Street, 1, Moscow 115478, Russia. Tel.: +7 4996128193; fax: +7 4993240702. E-mail address: avePlato@mail.ru (A.V. Ermakov). phenomenon were proposed, including transmission of signals by direct cellular contact and interaction through intercellular gap- junctions or through the culture medium of irradiated cells [11–15]. Many candidate molecules, including proteins, were proposed to the role of stress signaling factors [11,12]. In our previous study we showed that an extracellular DNA (ecDNA) derived from the cell genome participates in the development of BE induced by X-ray exposure in human G 0 -lymphocytes and human umbilical vein epithelial cells (HUVECs) [7,16]. We postulated that radiation- sensitive cells undergoing apoptosis serve as a source of ecDNA fragments that diffuse in the medium and bind to DNA-recognizing receptors on the surface of the bystander cells. According to our observations, BE could be stimulated by ecDNA of irradiated cells, but not by ecDNA produced by normal cells elim- inated by apoptosis. In this study, we tested the hypothesis that the difference between these two types of ecDNA is explained by DNA oxidation events occurring during and after irradiation. We compared the production of the nitric oxide and the ROS in human endothelial cells that were (1) irradiated at a low dose; (2) exposed to the ecDNA extracted from the media conditioned by irradiated cells; and (3) exposed to the genomic DNA oxidized in vitro. We found that the cellular responses to all three stimuli described above are essentially similar. We conclude that irradiation-related oxidation of the genomic DNA is an important component of the ecDNA-mediated bystander effect. 0027-5107/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2011.09.005