Sensitivity of mammalian cells to higher concentrations of reactive oxygen species induced by radiation or chemical treatment The reactive oxygen species (ROS) are group of very unstable compounds that can react with almost anything. This study is aimed to their reaction with biological objects, especially the mammalian cells. The ROS incorporate various different compounds [1]: - hydroxyl radical (OH ) - peroxyl (ROO ) and hydroperoxyl (HOO ) radicals - various peroxides (ROOR ) and hydroperoxides (ROOH) - singlet oxygen ( 1 O2) - superoxide anion ( O2 ) - hypochlorous acid (HOCl) - nitric oxide (NO) - peroxonitrite anion (ONOO ) Reactive oxygen species can be encountered, in low concentrations, basically in every living cell under normal conditions the ROS are part of the standard cellular metabolism. Their concentration can quickly rise when the cells are exposed to some abnormal environment, such as: hyperoxia, hyperthermia, hypooxia, chemical compounds (e.g.: hydrogen peroxide, menadione, etc.). Another example of abnormal environment is the exposure to ionizing radiation. The radiation can damage living organisms and their DNA, which is believed to be the critical target, in direct or Fig. 1 Comparison of the direct and indirect effect of radiation - illustration of double strand break (DSB) resulting from ionization within a single track. [2]
indirect manner. The reactive oxygen species are considered to be one of the most damaging agents causing the indirect effects of radiation - Fig. 1 [2]. Under normal conditions, the cells are well prepared to inactivate the created excessive ROS. For example, superoxide anion can be generated as by product from the oxygen during the mitochondrial respiration. This superoxide is metabolized by a family of enzymes superoxide dismutases to oxygen and hydrogen peroxide. Subsequently, the hydrogen peroxide may be turned by catalase to hydroxyl radical and afterwards to water - Error! Reference source not found.. This process of creation and transformation of ROS is based on the addition of electrons. However, during irradiation this process is reversed ionizing radiation is causing the ionization and excitation of water, leading to the removal of electrons. The majority of the ROS formed during irradiation come from the radiolysis of water [3]. Concentrations of ROS can be greatly elevated (in comparison to the normal conditions); the cell s natural antioxidant defenses overwhelmed, causing the oxidative stress within the cells and potential damage. The research of ROS is very up-to-date, even one of the hypotheses for the cause of genomic instability is involving the long-term elevated concentrations of ROS (e.g. after irradiation). There are still many uncertainties connected to the effects and persistency of the ROS in cells and a further research is essential. Fig. 2 - Formation and elimination of ROS under standard conditions and after exposure to ionizing radiation.
References 1. Myhre, O.; Andersen, J.M.; Aarnes, H.; Fonnum, F. Evaluation of the probes 2',7' dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochemical Pharmacology, 2003, vol. 65, p. 1575-1582. 2. Goodhead. D. T. Mechanisms for the biological effectiveness of high-let radiations. Journal of Radiation Research, 1999, vol. 40 (Suppl.), p. 1 13. 3. Yamamori, T.; Yasui, H.; Yamazumi, M.; et al. Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint. Free Radical Biology and Medicine, 2012, vol. 53, p. 260-270. Student activities and tasks - Acquaintance with the basic procedures of handling the cell cultures - Preparation of samples for irradiation or chemical treatment - Analysis of the ROS concentrations in cells (both in suspension and adhered to the surface) - Discussion of the obtained results and preparation of the final presentation During this course, participants will be given short basic lectures on radiobiology, focused mainly on: general cytological principles, radiation damage to DNA, differences between low LET and high-let radiation, role of ROS in organism, and genomic instability. Students will get familiar with the basics of the work in the biological laboratory. They will learn the proper handling of mammalian cell culture (Chinese hamster cells, line V79; Fig. 3) which involves: maintaining the cell culture, recultivation, counting the Fig. 3 Work with the cell culture under sterile conditions in the laminar flow hood.
concentration of cells in the suspension, distinguishing the dead cells from the living ones (trypan blue assay or colony formation assay), sample preparing for the irradiation, etc. This project is focused primarily on the measurement of reactive oxygen species in cells after irradiation and after chemical treatment. The concentrations of ROS will be measured immediately after the treatment as well as after prolonged periods of time. The experiments will be conducted on cells adhered to a surface as well as on the cells in suspension both approaches have their Fig. 4 Rokus M irradiatior ( 60 Co). advantages and disadvantages that should be clarified during the practice. The Joint Institute for Nuclear Research, Dubna (JINR) has plenty of possibilities for irradiation. There is gamma irradiator Rokus M ( 60 Co; Fig. 4), Phasotron for proton acceleration (up to 660 MeV/p + ), Synchrotron accelerator Nuclotron and U-400M cyclotron for heavy ion irradiation, and others. The type of radiation will be used according to the availability at the time of the summer practice. Another option for the ROS induction is the addition of chemical substances, such as: hydrogen peroxide, menadione, etc. The ROS in cells will be measured using the general oxidative stress indicator CM H2DCFDA (Life Technologies; C6827; Fig. 5, 1)) which is a fluorescent dye from the dichluorofluorescein (DCF) family of compounds. The DCF passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases (Fig. 5, 2)) and its thiol-reactive Fig. 5 - CM H2DCFDA fluorescent dye: 1) standard form; 2) acetate groups are cleaved by intracellular esterases; 3) oxidized, fluorescent dye.
chloromethyl group reacts with intracellular glutathione and other thiols. Subsequent oxidation yields a fluorescent adduct that is trapped inside the cell and can be used for long-term measurements Fig. 5, 3). Oxidized, fluorescent dye can be easily measured spectroscopically, using the microplate reader (BioTek Synergy H1 Multi- Mode Reader; Fig. 6) for adhered cells or Qubit 2.0 fluorometer (Life Technologies; Fig. 7) for cells in suspension. After completion, the students should be able to recultivate the V79 cells, prepare samples for chemical treatment and/or irradiation, apply the kit for ROS measurements, measure the levels of ROS both in suspension and in the adhered cells, and perform basic evaluation of the obtained data. Fig. 6 - Multi-Mode Reader; BioTek Synergy H1. Requirements - Basics of the work in chemical/biological laboratory (e.g. use of the automatic pipettes) - Basic knowledge of biology and the interaction of ionizing radiation with matter are welcomed Number of students This project is prepared for 1 student and his/her intensive learning of the subject. Practical part will take place after the introductory lessons and general explanation of the problematic. Recommended literature 1. Hall, E. J; Giaccia, A. Radiobiology for the radiologist. 6th ed. Philadelphia: Lippincott Williams & Wilkins, 2006 2. Woolley, J.F.; Stanicka, J.; Cotter, T.G. Recent advances in reactive oxygen species measurement in biological systems. Trends in Biochemical Sciences, 2013, vol. 38 (11), p. 556-565 Fig. 7 - Fluorometer Qubit 2.0; Life Technologies.
3. Hancock, J.T.; Desikan, R.; Neill, S.J. Role of Reactive Oxygen Species in Cell Signaling Pathways. Biochemical and Biomedical Aspects of Oxidative Modification, 2001, vol. 29 (2), p. 345-350 Project supervisor Pavel Bláha, Research Scientist, pavel.blahax@gmail.com Project co-supervisors Igor V. Koshlan, Scientific Secretary of Laboratory of Radiation Biology, koshlan@jinr.ru Natalia A. Koshlan, Research Scientist, nkoshlan@jinr.ru Group of Radiation Cytogenetics, Laboratory of Radiation Biology, JINR