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RADIOBIOLOGY 99 DNA DAMAGE IN SUBFRACTIONS OF HUMAN LYMPHOCYTES IRRADIATED WITH LOW DOSES OF X-RADIATION Marcin Kruszewski 1,2/ , Maria Wojewódzka 1/ , Teresa Iwaneńko 1/ , Aneta Goździk 3/ , Tomasz Ołdak 2,3/ , Eugeniusz K. Machaj 2,3/ , Zygmunt Pojda 2,3/ 1/ Institute of Nuclear Chemistry and Technology, Warszawa, Poland 2/ The Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Warszawa, Poland 3/ Military Institute of Hygiene and Epidemiology, Warszawa, Poland The aim of this study was to find a reliable, sensitive, fast and relatively cheap method to estimate radiation-induced DNA damage in human lymphocytes during a short time period after irradiation. We compared three methods: comet assay, micronucleus test and formation of phosphorylated histone H2AX foci. Human peripheral blood lymphocytes were collected by vein puncture and fractionated using T Cell Isolation Kit II and B Cell Isolation Kit II (Miltenyi Biotec), according to the manufacturer recommendations. Homogeneity of both T and B cell populations was 97-99%, as estimated by flow cytometry. Cells were irradiated with low doses of X-rays (0-1 Gy, 180 kV, 18 mA, 1 mm Cu filter) Fig.1. Microscopic image of nuclei from X-irradiated (1 Gy) human lymphocytes with repair foci visualised with anti-H2AX histone monoclonal antibody. using Stabilipan X-ray machine (Siemens). Dose rate was 1.2 Gy/min. DNA damage was assessed by standard alkaline comet assay [1], micronucleus test [2] and from the number of phosphorylated histone H2AX foci estimated by immunofluorescence [3] (Figs.1 and 2). The results indicate that all tests have similar sensitivity, approximately 0.3 Gy. All methods also reveal slightly higher induction of DNA damage in B lymphocytes as compared with T cells. The highest ratio of damage marker in irradiated cells (1 Gy) to control cells was obtained for formation of γH2AX foci; it was equal to 8.3, whereas for comet assay and micronucleus test, it was 4.0 and Fig.2. Formation of phosphorylated histone H2AX foci upon irradiation (a) and repair of DNA damage (b) induced by 1 Gy of X-rays, reflected as disappearance of phosphorylated histone H2AX foci. 4.1, respectively. The repair of DNA estimated by the γH2AX foci scoring was much slower as compared to the repair of total DNA damage measured by the comet assay. In summary, our study indicates that γH2AX foci assay is an advantageous alternative for micronucleus test and comet assay for radiation dose estimation in irradiated individuals. Long time of repair of DNA damage and high irradiated/control ratio make this assay more convenient than the other two methods applied. The work was supported by the Polish Ministry of Scientific Research and Information Technology statutory grant for the INCT. References [1]. Wojewódzka M., Kruszewski M., Iwaneńko T., Collins A.R., Szumiel I.: Mutat. Res., 416, 21-35 (1998). [2]. Wojewódzka M., Walicka M., Sochanowicz B., Szumiel I.: Int. J. Radiat. Biol., 66, 99-109 (1994). [3]. Cowell G., Durkacz B.W., Tilby M.J.: Biochem. Pharmacol. (2006), in press.
100 RADIOBIOLOGY THE ROLE OF LYSOSOMAL IRON IN NITRIC OXIDE SIGNALLING Sylwia Męczyńska, Hanna Lewandowska-Siwkiewicz, Marcin Kruszewski A considerable amount of cellular iron remains in the form of complexes with low molecular mass ligands, labile enough to enter the Fenton reaction. This pool has been called the labile iron pool (LIP). It plays a role, among others, in cellulariron transport, expression of iron regulatory genes, control of the activity of iron containing proteins, and catalysis of the Fenton reactions. It was proposed (e.g. [1,2]) that a part of the cellular pool of labile iron is confined within the acidic vacuolar compartment (lysosomes). Our previous results [3] show that LIP plays a role in the regulation of nitric oxide-dependent biochemical pathways, forming dinitrosyl iron complexes (DNIC), a group of physiologically important transducers of nitric oxide. Formation of nitrosyl iron complexes, known as 2.03 complexes due to the g value of their characteristic EPR spectra, has been found by several groups of researchers in many kinds of bacteria, plants and animals. It is postulated that DNIC are important factors Fig.1. EPR (electron paramagnetic resonance) signal intensity in K-562 cells incubated with DFO prior to the treatment with nitric oxide donor. Cells were treated with 1 mM DFO and incubated at 37 o C for 1, 3 or 6 h. After that time, nitric oxide donor was added to generate DNIC. in nitric oxide-dependent regulation pathways in the cell(e.g. [4,5]). The low molecular-weight DNIC have been shown to modulate redox properties of the cellular interior through the inhibition of glutathione-dependent enzymes, such as glutathione reductase, transferase and peroxidase. The sources of iron forming DNIC in vivo are still not precisely defined, one of the putative sources being the LIP and another – iron proteins. Neither are defined the cellular compartments, in which DNIC are formed. In the present <strong>report</strong>, we show that depletion of lysosomal LIP by either chelation with deferoxamine (DFO) or lysis inhibition (by treatment with 10 mM ammonium chloride) in K-562, human Fig.2. EPR signal intensity in K-562 cells incubated with ammonium chloride prior to the treatment with nitric oxide donor. Cells were treated with 10 mM ammonium chloride and incubated at 37 o C for 2, 4 or 6 h. After that time, nitric oxide donor was added to generate DNIC. myelogenous leukemia cells leads to a considerable decrease (down to 50%, depending on the incubation time) of DNIC forming in the cells treated with 70 µM nitric oxide donor (DEANO). The EPR spectra were recorded on Bruker ESP 300 at 77 K, microwave power – 1 mW, microwave frequency – 9.31 GHz, modulation amplitude – 3.027 G and time constant – 41 ms. In order to estimate the values of g coefficients, computer simulation was performed on SimFonia 1.25 software (Bruker Analytische Messtechnik, DE). The results presented in Figs.1 and 2 indicate a vital role of lysosomal labile iron in DNIC formation. Taken together, our present and previous results are consistent with the thesis on the considerable contribution of lysosomal iron to the total LIP in the cell. The work was supported by the Polish Ministry of Scientific Research and Information Technology statutory grant for the INCT. References [1]. Kurz T., Leake A., von Zglinicki T., Brunk U.T.: Ann. N. Y. Acad. Sci., 1019, 285-288 (2004). [2]. Persson H.L., Kurz T., Eaton J.W., Brunk U.T.: Biochem. J., 389(Pt 3), 877-884 (<strong>2005</strong>). [3]. Kruszewski M., Lewandowska H., Starzyński R.R., Bartłomiejczyk T., Iwaneńko T., Drapier J.-C., Lipiński P.: The role of labile iron pool and dinitrosyl iron complexes in nitric oxide genotoxicity. Summer Meeting SFRR – Europe 2004 “Reactive oxygen species and antioxidants”, Łódź, Poland, 02-05.07.2004. [4]. Rogers P.A., Eide L., Klungland A., Ding H.: DNA Repair, 2, 809-817 (2003). [5]. Turella P., Pedersen J.Z., Caccuri A.M., De Maria F., Mastroberardino P., Lo Bello M., Federici G., Ricci G.: J. Biol. Chem., 278, 42294-42299 (2003). RADIOSENSITIVITY OF HUMAN CHROMOSOMES 2, 8 AND 14. PART 1. PRIMARY BREAKS Sylwester Sommer, Iwona Buraczewska, Maria Wojewódzka, Irena Szumiel, Andrzej Wójcik A number of studies have been performed with the aim of verifying if the inter-chromosomal distribution of radiation-induced aberrations is proportional to the DNA content of the chromosome (reviewed in [1,2]). While deviations from randomness have been observed for a number of chromosomes, no consensus exists as to which chromosomes are repeatedly more or less sensitive than
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ANNUAL REPORT 2005 50 years in the
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CONTENTS GENERAL INFORMATION 9 MANA
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DETERMINATION OF CADMIUM, LEAD, COP
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NUCLEONIC CONTROL SYSTEMS AND ACCEL
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GENERAL INFORMATION 9 GENERAL INFOR
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MANAGEMENT OF THE INSTITUTE 11 MANA
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MANAGEMENT OF THE INSTITUTE 13 •
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SCIENTIFIC STAFF 15 5. Danilczuk Ma
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NUKLEONIKA 169 NUKLEONIKA THE INTER
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NUKLEONIKA 171 7. Influence of time
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INTERVIEWS IN 2005 173 INTERVIEWS I
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EDUCATION 209 EDUCATION Ph.D. PROGR
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RESEARCH PROJECTS AND CONTRACTS 211
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RESEARCH PROJECTS AND CONTRACTS IAE
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LIST OF VISITORS TO THE INCT IN 200
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LECTURES AND SEMINARS DELIVERED OUT
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LECTURES AND SEMINARS DELIVERED OUT
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AWARDS IN 2005 221 AWARDS IN 2005 1
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INDEX OF THE AUTHORS 235 Lisowska H
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