This is the accepted version of the journal article: Verdejo, Valentina; Radl, Analía; Barquinero, Joan Francesc; [et al.]. «Use of a plasma focus device to study pulsed x-ray effects on peripheral blood lymphocytes : Analysis of chromosome aberrations». Journal of applied physics, Vol. 133 (April 2023), art. 163302. DOI 10.1063/5.0141529 This version is available at https://ddd.uab.cat/record/274290 under the terms of the license This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 1 Use of a Plasma Focus Device to study pulsed X-rays effects on Peripheral Blood 2 Lymphocytes: Analysis of Chromosome Aberrations 3 4 Authors: Valentina Verdejoa, Analía Radla, Joan-Francesc Barquinerob, Jalaj Jainc, Sergio 5 Davisc,d, Cristian Pavezc,d, Leopoldo Sotoc,d and José Morenoc,d* 6 7 Affiliations: a 8 Laboratorio Dosimetría Citogenética, Comisión Chilena de Energía Nuclear, Casilla PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 9 188-D, 7600713 Santiago, Chile b 10 Departamento de Biología Animal, Biología Vegetal y Ecología, Universitat Autònoma 11 de Barcelona, 08193 Barcelona, Spain c 12 Research Center on the Intersection in Physics of Plasmas, Matter and Complexity, 13 P2mc, Comisión Chilena de Energía Nuclear, Casilla 188-D, 7600713 Santiago, Chile d 14 Universidad Andrés Bello, Departamento de Ciencias Físicas, Republica 220, Santiago- 15 8370035, Chile 16 17 *Corresponding author: José Moreno. E:mail: jose.moreno@cchen.cl. Phone: +562 2364 18 6243 1 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 19 ABSTRACT 20 X-ray pulses (Full Width at Half Maximum ~ 90 ns, dose rate ~ 107 Gy·sec-1) were used 21 to irradiate a monolayer of peripheral blood mononucleated cells (PBMCs) using the PF- 22 2kJ kilojoule plasma focus device. Four different exposure conditions were evaluated 23 using 5, 10, 20, and 40 pulses, with the mean dose measured by TLD-100 being 0.12 24 ±0.02 mGy, 0.14±0.03 mGy, 0.22±0.06 mGy, and 0.47±0.09 mGy, respectively. 25 Cytogenetic analysis showed an increase in all types of chromosomal aberrations 26 following exposure to X-ray pulses. PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 27 The distribution of dicentrics and centric rings was overdispersed after 5, 10, 20 and 40 28 pulses. Additionally, after 20 and 40 pulses the presence of tricentric chromosomes is 29 detected. Chromosome aberration frequencies found in this study were always higher than 30 the estimated frequencies of chromosome aberrations using published dose effect curves 31 for conventional radiation sources. The overdispersion observed, the elevated Maximum 32 Relative Biological Effectiveness, RBEM, and the presence of tricentric chromosomes at 33 the relatively low doses of exposure (<0.5 Gy) seems to indicate that low doses of pulsed 34 X-rays of low energy show similar biological effects as those observed for high-LET 35 radiation. X-rays pulses emitted by PF- 2kJ were found more efficient in inducing 36 chromosome aberrations, even more than α particles. 37 38 39 Keywords: Chromosome aberrations, pulsed X-rays, peripheral blood lymphocytes, low 40 dose, plasma focus device 41 2 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 42 1. Introduction 43 44 The development and use of pulsed radiation sources in different fields of science and 45 industry [1-2], make it necessary to know and characterize the effects of these radiations 46 on different types of matters. The evaluation of their biological effect is of relevance for 47 their possible applications in radiotherapy treatments, and for radiological protection 48 measures. 49 Several studies showed that the ultra-high dose-rate (UHDR) pulsed (Full Width at Half 50 Maximum, FWHM of tens of nanoseconds, ~109 Gy·s-1) irradiations of high energies (~ PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 51 MeV) effects on biological samples are not so different from continuous-conventional 52 (CONV) irradiation effects [3-5]. Recently, pulsed X-ray emitted from a kilojoule plasma 53 focus device, PF-2kJ (FWHM of about 90 ns, 107 Gy·s-1, low energy 8-10 keV), has been 54 applied to irradiate several cancer cell lines and the obtained results showed a higher cell 55 death in comparison to conventional X-ray source irradiation at the same doses [6]. In 56 other studies, using plasma focus devices, the higher effects of pulsed X-rays irradiation 57 on cancer cells have been reported [7-9]. The above mentioned research demand to 58 continue the study in order to understand the difference on the biological effect between 59 pulsed and conventional (continuous) radiation, analyzing different biological endpoints 60 (cell survival, mutations and chromosome aberrations) and/or cell lines (tumor cell, stem 61 cells, blood cells). To do so in the present work, a kilojoule plasma focus device, PF-2kJ, 62 was used to irradiate blood lymphocyte samples. The chromosome aberrations from 63 peripheral blood lymphocytes were analyzed, this cytogenetic biomarker is widely used 64 in radiobiology and radioprotection for conventional radiation sources. Since a plasma 65 focus device is used in the present work, in the following a brief of radiation emission 66 from plasma focus devices is presented. 3 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 67 Plasma focus devices produce pulsed plasma and radiation pulses using transient 68 electrical discharges. Various types of pulsed radiation are emitted from the plasma focus 69 devices (~10-100 ns); low (~ 5-15 keV) and high-energy (> hundreds of keV X-rays) [10- 70 15], neutrons (~ MeV) [11, 16-19], ions [11, 20-23], relativistic electrons [24], and ultra- 71 high frequency (UHF) electromagnetic radiation [25-27]. Various schemes have been 72 proposed to improve the performance of plasma focus devices concerning radiation 73 emission [28-30]. Different electrode geometries; oval-shaped anode, conical top anode, 74 and stepped anode have been tested and it was found that the conical top anode had a PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 75 better performance [29-30]. The present work uses a conical top anode to get the 76 maximum X-ray emission using hydrogen as the working gas that only produces X-rays. 77 Among other effects induced by ionizing radiation (IR), like X-rays, the double-strand 78 breaks in the DNA molecule are the most important. Consequences of these lesions are 79 mutations and chromosome aberrations due to unrepair or misrepair during the cellular 80 cycle division [31]. Dicentric chromosomes are formed by the misjoining of two broken 81 chromosomes that carry the centromeric region of each chromosome; the formation of 82 centric rings results from the erroneous joining of a chromosome that breaks into two 83 arms, joining itself. In either of these cases the remaining acentric chromosome fragments 84 also join, forming what is known as an acentric fragment [32]. These chromosome 85 aberrations (CA) are almost exclusively induced by IR [33]. The CA frequency is also 86 commonly used as a cytogenetic biomarker of dose exposure [34]. The analysis of this 87 biomarker in peripheral blood lymphocytes (PBL) is considered a robust and “gold 88 standard” biological dosimetry method and an important tool in the area of radiation 89 protection [35-36]. Dicentric chromosomes and centric rings are the most reliable and 90 repeatable method for comparing biological response for a wide range of doses and 91 qualities of ionizing radiation [37]. 4 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 92 The Relative Biological Effectiveness (RBE) is the ratio of the absorbed doses of two 93 types of radiation that produce the same specified effect, and the Maximum Relative 94 Biological Effectiveness (RBEM) is the ratio of linear coefficients (α coefficient) of the 95 dose–response curves for the radiation of interest and a reference radiation [38]. The 96 RBEM using chromosome aberrations as cytogenetic endpoint in PBL after IR exposure 97 has been studied for different radiation qualities [37, 39-41]. High linear energy-transfer 98 (high-LET) irradiations, such as neutrons and α-particles, have greater biological 99 effectiveness than low linear energy-transfer (low-LET), like X-rays or γ-rays. However, PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 100 low-energy X-rays are more biologically effective, per unit absorbed dose, than high- 101 energy X-rays or γ-rays due to the production of lower energy secondary electrons [37, 102 40, 42-43]. RBEM values have not been reported for low-dose, ultra-high dose rate and 103 low-energy pulsed X-rays. Thus, in the present work a kilojoule plasma focus device, PF- 104 2kJ, is adapted as an ultra-high dose rate pulsed low-energy X-rays source (8-10 keV, 105 FWHM ~90 ns, 107 Gy·s-1), to evaluate the biological effectiveness using chromosome 106 aberrations as cytogenetic biomarker in peripheral blood lymphocytes (PBL). 107 108 2. Methods 109 2.1. Experimental Setup. 110 A schematic of the kilojoule plasma focus device, PF-2kJ [23], for dose measurement and 111 lymphocyte irradiation is shown in Figure 1. PF-2kJ (Figure 1-A) consists of a central 112 electrode, anode, partially covered by an alumina insulator. The conical anode top 113 geometry was proposed to have better pinching action in a PF device with ~ 2kJ energy 114 [29], thus being used in the present work. Normally, the PF devices consist of a coaxial 115 electrode geometry in which cathode bars symmetrically surround the central anode, 116 unlike the present work. High X-ray emission is reported for the same device and others 5 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 117 without cathode bars [30,44]. The external diameter of the anode was ~24 mm and the 118 effective length was ~ 46 mm. Electrical parameter of the PF-2kJ can be found in Jain et 119 al. 2019 [23]. A photomultiplier tube, PMT, model Photonis XP2262B (Photonis Imaging 120 sensors, USA) in combination with the scintillator BC-408 (Saint Gobain, USA) was 121 mounted at a distance of ~ 82 cm in the axial direction to monitored the presence of X- 122 ray as reference tool. A lead piece was kept at the bottom of the anode to increase the X- 123 ray emission [7]. An array of 21 TLD-100 thermoluminescent dosimeters (RadPro, 124 Wermelskirchen, Germany) was arranged in a petri dish (Figure 1-B) and kept over the PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 125 plastic vacuum window at a distance of ~5.5 cm from the anode top. These dosimeters 126 show linearity in the energy range keV to MeV [45-47]. 127 Plasma focus devices emit a large amount of X-ray in the low energy range [48]. The 128 estimated average X-ray energies (low-energy zone) emitted from the PF-2kJ are about 129 8-10 keV. Such estimations were based on the dosimetric measurements and HVL values 130 [49]. Because of this, a high-density plastic vacuum window was used so that maximum 131 X-ray transmission can take place [7]. 6 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 132 133 Figure 1. Experimental setup. (A) Schematic of the kilojoule plasma focus device, PF- 134 2kJ, PMT: photomultiplier tube; VDR: resistive voltage divider, SG: Spark-Gap, RC: 135 Rogowski coil, CB: capacitor bank (B) arrangement of TLD-100 dosimeters, and (C) 136 arrangement for cell irradiation. 137 138 139 2.2. Isolation of Peripheral Blood Mononucleated Cells. 140 Peripheral blood samples from a 50-years-old male with no ionizing radiation or 141 clastogenic exposure history were collected in heparinized tubes. Previous informed 142 consent was obtained. The same blood donor has been used in all irradiation conditions, 143 so that the influence of interindividual variations is eliminated. 144 Because of the attenuation of photon energies (8-10 keV) of this plasma focus device, 145 irradiation assays were performed using a monolayer of human peripheral lymphocytes. 146 For this, peripheral blood mononucleated cells (PBMCs) were separated using the density 147 gradient method by Histopaque® 1077 (Sigma-Aldrich Company Ltd., Gillingham, 148 United Kingdom). Three milliliters of heparinized blood were mixed 1:1 in RPMI-1640 7 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 149 (supplemented with L-glutamine and 25 mM HEPES; Gibco, Grand Island, New York, 150 USA), gentled deposited onto 3 mL of Histopaque-1077 and centrifuged at 400 ×g for 30 151 min at room temperature. After aspirating the upper layer, the opaque interface containing 152 PBMCs was collected into a clean 15 mL conical tube (Corning Inc., New York, USA). 153 Cells were washed using 10 mL of RPMI-1640 medium, after centrifuging at 700 ×g for 154 15 min, the supernatant was discarded and cells were resuspended on 2 mL of RPMI- 155 1640. Using a dilution of 1:10 of this cell solution, concentration was calculated by 156 Neubauer chamber (Brand, Wertheim, Germany). PBMCs were diluted considering the PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 157 maximum cell concentration to achieve a monocellular layer of cells on a 35 mm Petri 158 dish that is 6.13·106 cell·mL-1. In all irradiation conditions 2 mL of cell solution was 159 added to Petri dish. 160 161 2.3. Irradiation Conditions 162 Irradiation assays were done on 35 mm polystyrene Petri dishes, uncoated and tissue 163 culture treated (Nest, Jiangsu, China) (Figure 1-C). After the addition of the PBMCs 164 dilution, the dishes were left undisturbed for 30 min to allow sedimentation according to 165 the method of Virsik et al. [50]. The monolayers of cells settled on the bottom of the 166 dishes were exposed separately to 5, 10, 20, and 40 pulses of X-ray emitted from the PF- 167 2kJ device (FWHM ~ 90 ns, dose rate ~ 107 Gy·s-1), two independent experiments were 168 performed for each irradiation conditions. Additionally, an unirradiated monolayer 169 sample was prepared to evaluate the effect of sham irradiated conditions. An array of 21 170 TLD-100 dosimeters in the same type of Petri dish was positioned at the anode top, to 171 measure the doses under the same irradiation conditions. 172 173 2.4. Lymphocyte Culture and Chromosome Analysis 8 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 174 After irradiation, cells were collected in a 15 mL conical tube (Corning), washed with 2 175 mL of RPMI 1640 medium, and centrifuged 10 min to 700 ×g. The supernatant was 176 discarded and cells were resuspended on 0.5 mL of RPMI 1640 medium. The cells were 177 incubated at 37 °C for 2 h with 4 mL RPMI 1640 medium, supplemented with 18 % of 178 Fetal Bovine Serum (Gibco, Grand Island, New York, USA). To stimulate lymphocyte 179 growth Phytohemagglutinin M (Gibco, Grand Island, New York, USA) 1.8 % v/v was 180 added. Lymphocyte cultures were done in presence of 5-Bromo-2´-deoxyuridine (0.9 % 181 v/v) (Calbiochem, San Diego, California, USA). Since an increase in radiation dose can PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 182 delay the progression of the cell cycle [51-53], and in order to obtain a sufficient number 183 of cells for analysis, the length of culture were 48 h, 50h and 72h for 5-10, 20 and 40 184 pulses respectively. In all the cases, Colcemid (Gibco, Grand Island, New York, USA) 185 was added after 45 h of culture to obtain a final concentration of 0.1 μg·mL-1 of Colcemid. 186 All cultures were incubated at 37 °C. After incubation, cultures were centrifuged for 10 187 min 700 g and the supernatant was replaced by hypotonic solution (KCl 0.075 M, Gibco, 188 Grand Island, New York, USA) prewarmed at 37 °C. After 10 minutes of treatment with 189 the hypotonic solution at 37 °C cultures were centrifuged 5 min 700 ×g, and cells were 190 fixed with methanol and acetic acid (3:1). For the cytogenetic analysis 2 to 3 days old 191 slides were stained by the fluorescence plus Giemsa technique, using Hoechst 33258 stain 192 in pH 6.8 phosphate buffer. 193 Chromosome analysis was carried out exclusively on first division metaphases containing 194 46 centromeres. The dicentric chromosomes (dic), tricentric chromosomes (tric), and 195 centric rings (r) were only considered when their corresponding acentric fragment was 196 present. Acentric fragments not related to a multicentric or a ring chromosome were 197 recorded as extra acentric fragments (ace). For each irradiation condition, and following 198 international criteria in cytogenetics dosimetry [34], the number of metaphases analyzed 9 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 199 was approximately 500, or those needed to score 100 chromosome aberrations. All 200 metaphases with chromosome aberrations were analyzed independently by two scorers. 201 During the analysis, the mitotic index was also calculated [34]. 202 203 2.4. Statistical Analysis 204 To check if the distribution of chromosome aberrations after each irradiation condition 205 followed a Poisson distribution, the dispersion index, variance/mean, and the normalized 206 unit of this index, the U-test, were used [54]. Differences among irradiation conditions PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 207 were evaluated by a one-way ANOVA test. Ordinary least squares method was used to 208 calculate the linear regression between the dose and the frequency of chromosome 209 aberrations. 210 211 3. Results 212 The average dose (±SD) of TLD measurements at 5, 10, 20 and 40 pulses are 0.12 (±0.02) 213 Gy, 0.14 (±0.03) Gy, 0.22 (±0.06) Gy and 0.47 (±0.09) Gy respectively. From 5 to 10 214 pulses there was a slight non-significant increase in the mean dose, but from 10 to 40 215 pulses there was a linear increase in the mean dose (Figure 2). The slope of the linear 216 regression indicates a mean of 12 mGy per pulse. 10 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 217 PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 218 Figure 2. Mean dose measurement using TLD-100 in four irradiation conditions (5, 10, 219 20, and 40 pulses), bars represent standard deviation and broken lines represent linear fit. 220 221 Figure 3 shows microphotographs of representative metaphase for different X-rays pulsed 222 conditions. Cytogenetic results are summarized in Table I. Eight out of 10 total irradiated 223 samples could be analyzed to achieve ~ 500 cells or 100 dicentrics plus rings (40 pulses 224 irradiation condition), and only in two cases, the number of cells analyzed was between 225 400 and 450 (at 5 and 20 pulses). The mitotic index was always higher than 2.5 %, which 226 is considered a reference value in radiation cytogenetics [34]. In this work, the mitotic 227 index seems not to be influenced by the number of X-rays pulses. One-way analysis of 228 variance (ANOVA) is used to determine statistical significance differences between the 229 chromosomal aberrations scoring on different exposure conditions, for dicentrics and 230 extra acentric fragments, there was a clear increase as the number of pulses increased (p< 231 0.0001 in both cases). Significant differences were also observed for tricentric 232 chromosomes obtained after 20 and 40 X-rays pulses (p<0.0001), and centric rings 233 obtained for 5, 20, and 40 X-rays pulses (p<0.0002). The frequency of centric rings show 234 a tendency to increase with the number of pulses. When total chromosome aberrations 11 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 235 were considered, there was a clear increase (p < 0.001). When the replicas were 236 compared, similar frequencies of CA were observed. These results show the irradiation 237 methodology and experimental setup of PF2kJ are robust. 238 A B PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 239 Figure 3. Metaphase observed after 0 pulses (A), and after exposure to 10 X-rays pulses 240 (B). In B, the metaphase has one dicentric chromosome (blue arrow), one centric ring 241 (green arrow), and four acentric fragments (red arrow). 242 243 244 Table I Cytogenetic results obtained from lymphocytes irradiated with X-rays pulses. cells # pulses MI dic tric r ace Total CA scored 0 10.6 500 0 0 0 0 0 0 10 500 0 0 0 0 0 5 6.3 411 9 0 3 2 14 5 5.4 500 12 0 2 4 18 12 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 10 10.7 500 27 0 0 21 48 10 9.5 500 25 0 2 16 43 20 8.2 500 78 0 12 33 123 20 5.1 433 77 3 7 39 126 40 10.2 217 93 1 6 56 156 PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 40 8.2 225 91 1 7 50 149 245 MI: mitotic index; dic: dicentric chromosome; tric: tricentric chromosome; r: ring; ace: 246 acentric fragment; CA: chromosome aberrations 247 248 Figure 4 shows the frequencies of different chromosome aberrations, calculated from the 249 data in Table I, considering tricentrics as two dicentrics. As can be seen, for all types of 250 CA there is a clear increase with dose. The linear coefficients (α) were 1.298 (± 0.153), 251 0.861 (± 0.106), 0.802 (± 0.094), and 0.443 (± 0.062) for Total CA, dic+r, dic, and ace 252 respectively. 253 13 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 254 Figure 4. Dose dependence of chromosome aberrations in PBMCs irradiated by X-rays 255 from PF-2kJ. Blue triangles represent the frequency of total chromosome aberrations 256 (total CA); the red circles are dicentrics plus rings (dic+r); green diamond are dicentrics 257 (dic); and white hexagon are acentric fragments (ace). Broken lines represent linear fit. 258 259 The chromosome aberrations frequency was calculated as the ratio of dicentrics plus rings 260 and the total cells scored, Table II shows these results and dicentric plus ring cell 261 distribution. After uniform exposure to low-LET radiation, dicentric cell distribution 262 follows a Poisson distribution where the ratio between the variance and the mean, the PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 263 dispersion index (DI) tends to 1, the DI is higher than 1 in all cases, indicating a tendency 264 to show overdispersed values. This overdispersion was significant (U-test > 1.96) in one 265 replica of each irradiation condition. 266 267 Table II. Intercellular distribution of dicentric chromosomes plus centric rings after X- 268 rays pulses irradiation. dic+r distribution within cells cells dic+r # pulses DI U test scored scored 0 1 2 3 4 5 14 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 0 500 0 0 0 0 0 0 0 - - 0 500 0 0 0 0 0 0 0 - - 5 411 12 401 8 2 0 0 0 1.31 4.6 5 500 14 487 12 1 0 0 0 1.12 1.92 10 500 27 474 25 1 0 0 0 1.02 0.36 10 500 27 476 21 3 0 0 0 1.17 2.75 PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 20 500 90 418 75 6 1 0 0 1.02 0.35 20 433 90 361 55 16 1 0 0 1.22 3.21 40 217 101 147 46 22 2 0 1 1.30 3.09 40 225 100 151 51 20 3 0 0 1.14 1.50 269 dic+r: dicentrics plus rings; DI: Dispersion index (σ2/y); U test; normalized unit of 270 dispersion index, values >1.96 indicated overdispersion. 271 4. Discussion 272 The dicentric chromosomes and centric rings are two different kinds of unstable 273 chromosome aberrations, they are specific to ionizing radiation with a clear dependence 274 on dose, dose-rate and radiation quality. To the best of our knowledge, this work is the 275 first study where cytogenetic biomarkers are analyzed in order to evaluate the biological 276 effect of X-rays pulses at low doses (<0.5 Gy), ultra-high dose rate (107 Gy·s-1) and low 277 energies (8-10 keV). 278 A monolayer of peripheral mononucleated cells (PBMCs) settled on the bottom of a Petri 279 dish was used, in order to avoid a depth dose gradient in the irradiated sample due to the 280 strong attenuation of such low photon energies. Additionally, the irradiation of 281 unstimulated lymphocytes allows us to have all cells in the same phase of the cell cycle, 15 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 282 in this case in the G0 quiescent stage. According to the irradiation conditions presented in 283 [50, 55-58], to evaluate the induction of chromosome aberrations by conventional X-rays 284 of low energies, some important factors have to be taking into account: i) a monolayer of 285 cells, ii) cellular cycle control and iii) temperature control on irradiation assays. In the 286 present study, petri dishes were kept at 37 °C before and just after irradiation, and 287 although irradiations were not performed at 37 °C, they were above 17 °C as it is 288 suggested by Gumrich et al. [59]. 289 Previous studies have shown that higher doses of radiation result in an increased PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 290 frequency of chromosome aberrations and a delay in cell cycle [51-53]. Consequently, 291 the length of lymphocyte culture was set between 48-72 hours, depending on the number 292 of pulses. All mitotic indexes were above 5 %, indicating that culture results were 293 satisfactory. Because the cytogenetic analysis was restricted to first division metaphase, 294 underestimation due to the analysis of second or third division metaphases was avoided. 295 Radiation exposures at ultra-high dose rates (106-107 Gy·s-1, UHDRs) have been shown 296 to manifest differential radiobiological responses, and induced less damage, compared to 297 conventional (CONV) dose rates (0.001-0.4 Gy·s-1) [5]. The increase in radical–radical 298 recombination and oxygen depletion are the main hypotheses to explain a reduced yield 299 of biological lesions at ultra-high dose rates [60]. Previous cytogenetic studies using 300 UHDR have shown a decrease in the induction of chromosome aberrations when the 301 number of pulses or the dose rate increased [61-63]. It should be noted that these earlier 302 studies were performed in a higher energy range (in the order of MeV) and at higher 303 radiation doses (2-8 Gy). In contrast, in the present study, we observed an increase in 304 chromosome aberrations with the number of pulses. Our results seem to suggest that at 305 low doses and for low-energy X-rays delivered at UHDR, there is a major biological 306 effectiveness in producing DNA damage. Acharya et al. [63] observed an increase of 16 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 307 micronuclei yield when doses were delivered by multiple pulses compared with those 308 delivered by single pulse, especially at higher doses of 50 and 25 ns pulses. This seems 309 to indicate that for fractionation strategy short time of X-rays pulses (90 ns) which are 310 obtained from PF-2kJ, could be an important parameter on chromosome aberrations 311 induction. 312 Four reference dose-effect curves were applied to estimate the chromosome aberration 313 frequency expected at the physical doses reported in the present work (TLD-100): a α- 314 particle curve of 241Am 2.7 MeV, 0.1 Gy·min-1 [55]; two X-rays curves, one of 180 kVp, PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 315 0.27 Gy·min-1 [64], and another of 10 keV, 0.5 Gy·min-1 [42]; and one γ radiation curve 316 of 60Co, mean energy 1.25 MeV, 1.2-1.1 Gy·min-1 [65]. The frequencies values estimated 317 are shown in Figure 5, and compared with the frequency of dicentrics plus rings observed 318 in the present study. As it can be seen, the observed frequencies are higher than all the 319 frequencies that were estimated for the above indicated curves. 320 321 322 323 Figure 5. Comparison of dicentric plus rings (dic+r) frequency observed in the present 324 study (red circles), and frequencies calculated using previously published curves for α- 17 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 325 particles (green diamonds [55]), X-rays (blue invert triangle for 10 KeV [42]; and purple 326 cross for 180 kVp [64]), and γ radiation (yellow squares [65]). Broken lines represent 327 linear fit. 328 329 A weighted least-squares approximation was used to fit the data for each reference 330 radiation quality. Considering irradiation conditions were performed at low doses values 331 (< 0.5 Gy), and in order to compare the biological effect of X-rays pulses with 332 conventional ionizing radiations sources, RBEM was determined as the ratio α coefficient from linear fitting of the result reported in the present work, and α coefficient from linear PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 333 334 fitting for reference radiations quality (α particles, X-rays and γ radiation). RBEM values 335 are shown in Table III, these results indicate that the photon energy of pulsed X-rays 336 emitted by PF-2kJ is more effective compared even with high LET radiation. 337 Table III. Linear regression fit coefficient, and the biological relative effectiveness 338 (RBEM) for each type of radiation (α-particles, X-rays, γ-rays). Radiation α RBEM Present data 0.875±0.079 - α-particles[55] 0.288 ± 0.001 3.0 X-rays 10 keV[42] 0.181 ± 0.028 4.8 X-rays[64] 0.065± 0.004 13.5 γ-rays[65] 0.054 ± 0.004 16.2 339 α: linear coefficient; RBEM: relative biological effectiveness. 340 18 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 341 In this study, the results show effects as those that are expected after high LET radiation 342 exposures, indicating a different behavior of pulses radiation compared to conventional 343 radiation. The intercellular distribution of dic+r showed a significant overdispersion at 5, 344 10, 20, and 40 pulses, where the u-test were 4.6, 2.75, 3.21, and 3.09 respectively. Since 345 it is well known that for low-LET radiation dicentric and dicentric plus ring cell 346 distribution agrees with the Poisson distribution, where the variance is equal to the mean. 347 In the present study the overdispersion (variance > media) observed was unexpected. For 348 these chromosome type aberrations overdispersion is expected after non-homogeneous PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 349 exposure to low-LET radiation, or after high-LET radiation exposure. However, in this 350 work we observed tricentric chromosomes, these multicentric configurations are rarely 351 observed after low doses of low-LET exposure, and common after irradiations to low 352 doses of high-LET [66-67]. 353 Our results suggest that the pulsed X-rays of low-energy in low-dose range, with ultra- 354 high dose rate interact distinctively with the DNA. Considering PF-2kJ has these 355 characteristics it is necessary to gain a better understanding of mechanisms underlying 356 the DNA damage induced by X-rays pulses such as by analyzing the formation of 357 complex chromosome aberrations using fluorescence in situ hybridization (FISH) 358 techniques, or evaluating the repair kinetics of the double strain break by analyzing the γ- 359 H2AX foci [68]. The incorporation of others cytogenetic biomarkers could contribute to 360 the characterization, since they allow direct evidence of the effects of radiations in 361 biological systems. 362 363 5. Conclusions 364 In the present study our results evidence a different radiobiological response of PBMCs 365 to pulsed irradiation. The presence of tricentrics and the overdispersion observed at low 19 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. 366 doses, ultra-high dose rate and low energies of X-rays emitted by a PF-2kJ, suggest that 367 the biological effect of X-rays pulses seems to be like high LET. The RBEM analysis 368 confirms this observation, where the pulses emitted by PF-2kJ are more effective inducing 369 CA compared even with high LET radiation. 370 371 Acknowledgements 372 373 This work was financially supported by the ANID FONDECYT Regular project 1190677 374 Chile, the ANID FONDECYT Postdoctoral grant 3190184 Chile, and PIA Anillo PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 375 ACT172101 grant Chile. The authors are thankful to the department of dosimetry of 376 Chilean Nuclear Energy Commission for their support in the dose measurements. 377 378 Disclosure statement 379 No conflict of interest was reported by the authors. 20 This is the author’s peer reviewed, accepted manuscript. 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However, the online version of record will be different from this version once it has been copyedited and typeset. PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529 This is the author’s peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. PLEASE CITE THIS ARTICLE AS DOI: 10.1063/5.0141529