Use of a Plasma Focus Device to study pulsed X-rays effects on Peripheral Blood 1 Lymphocytes: Analysis of Chromosome Aberrations

19 X-ray pulses (Full Width at Half Maximum ~ 90 ns, dose rate ~ 10 7 Gy·sec -1 ) were used 20 to irradiate a monolayer of peripheral blood mononucleated cells (PBMCs) using the PF-21 2kJ kilojoule plasma focus device. Four different exposure conditions were evaluated 22 using 5, 10, 20, and 40 pulses, with the mean dose measured by TLD-100 being 0.12 23 ±0.02 mGy, 0.14±0.03 mGy, 0.22±0.06 mGy, and 0.47±0.09 mGy, respectively. 24 Cytogenetic analysis showed an increase in all types of chromosomal aberrations 25 following exposure to X-ray pulses. 26 The distribution of dicentrics and centric rings was overdispersed after 5, 10, 20 and 40 27 pulses. Additionally, after 20 and 40 pulses the presence of tricentric chromosomes is 28 detected. Chromosome aberration frequencies found in this study were always higher than 29 the estimated frequencies of chromosome aberrations using published dose effect curves 30 for conventional radiation sources. The overdispersion observed, the elevated Maximum 31 Relative Biological Effectiveness, RBE M , and the presence of tricentric chromosomes at 32 the relatively low doses of exposure (<0.5 Gy) seems to indicate that low doses of pulsed 33 X-rays of low energy show similar biological effects as those observed for high-LET 34 radiation. X-rays pulses emitted by PF-2kJ were found more efficient in inducing 35 chromosome aberrations, even more than α particles.


Introduction
The development and use of pulsed radiation sources in different fields of science and industry [1][2], make it necessary to know and characterize the effects of these radiations on different types of matters.The evaluation of their biological effect is of relevance for their possible applications in radiotherapy treatments, and for radiological protection measures.
Several studies showed that the ultra-high dose-rate (UHDR) pulsed (Full Width at Half Maximum, FWHM of tens of nanoseconds, ~10 9 Gy•s -1 ) irradiations of high energies (~ MeV) effects on biological samples are not so different from continuous-conventional (CONV) irradiation effects [3][4][5].Recently, pulsed X-ray emitted from a kilojoule plasma focus device, PF-2kJ (FWHM of about 90 ns, 10 7 Gy•s -1 , low energy 8-10 keV), has been applied to irradiate several cancer cell lines and the obtained results showed a higher cell death in comparison to conventional X-ray source irradiation at the same doses [6].In other studies, using plasma focus devices, the higher effects of pulsed X-rays irradiation on cancer cells have been reported [7][8][9].The above mentioned research demand to continue the study in order to understand the difference on the biological effect between pulsed and conventional (continuous) radiation, analyzing different biological endpoints (cell survival, mutations and chromosome aberrations) and/or cell lines (tumor cell, stem cells, blood cells).To do so in the present work, a kilojoule plasma focus device, PF-2kJ, was used to irradiate blood lymphocyte samples.The chromosome aberrations from peripheral blood lymphocytes were analyzed, this cytogenetic biomarker is widely used in radiobiology and radioprotection for conventional radiation sources.Since a plasma focus device is used in the present work, in the following a brief of radiation emission from plasma focus devices is presented.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 Plasma focus devices produce pulsed plasma and radiation pulses using transient electrical discharges.Various types of pulsed radiation are emitted from the plasma focus devices (~10-100 ns); low (~ 5-15 keV) and high-energy (> hundreds of keV X-rays) [10][11][12][13][14][15], neutrons (~ MeV) [11,[16][17][18][19], ions [11,[20][21][22][23], relativistic electrons [24], and ultrahigh frequency (UHF) electromagnetic radiation [25][26][27].Various schemes have been proposed to improve the performance of plasma focus devices concerning radiation emission [28][29][30].Different electrode geometries; oval-shaped anode, conical top anode, and stepped anode have been tested and it was found that the conical top anode had a better performance [29][30].The present work uses a conical top anode to get the maximum X-ray emission using hydrogen as the working gas that only produces X-rays.The Relative Biological Effectiveness (RBE) is the ratio of the absorbed doses of two types of radiation that produce the same specified effect, and the Maximum Relative Biological Effectiveness (RBEM) is the ratio of linear coefficients (α coefficient) of the dose-response curves for the radiation of interest and a reference radiation [38].The RBEM using chromosome aberrations as cytogenetic endpoint in PBL after IR exposure has been studied for different radiation qualities [37,[39][40][41].High linear energy-transfer (high-LET) irradiations, such as neutrons and α-particles, have greater biological effectiveness than low linear energy-transfer (low-LET), like X-rays or γ-rays.However, low-energy X-rays are more biologically effective, per unit absorbed dose, than highenergy X-rays or γ-rays due to the production of lower energy secondary electrons [37,40,[42][43].RBEM values have not been reported for low-dose, ultra-high dose rate and low-energy pulsed X-rays.Thus, in the present work a kilojoule plasma focus device, PF-2kJ, is adapted as an ultra-high dose rate pulsed low-energy X-rays source (8-10 keV, FWHM ~90 ns, 10 7 Gy•s -1 ), to evaluate the biological effectiveness using chromosome aberrations as cytogenetic biomarker in peripheral blood lymphocytes (PBL).

Experimental Setup.
A schematic of the kilojoule plasma focus device, PF-2kJ [23], for dose measurement and lymphocyte irradiation is shown in Figure 1.PF-2kJ (Figure 1

Isolation of Peripheral Blood Mononucleated Cells.
Peripheral blood samples from a 50-years-old male with no ionizing radiation or clastogenic exposure history were collected in heparinized tubes.Previous informed consent was obtained.The same blood donor has been used in all irradiation conditions, so that the influence of interindividual variations is eliminated.
Because of the attenuation of photon energies (8-10 keV) of this plasma focus device, irradiation assays were performed using a monolayer of human peripheral lymphocytes.
For this, peripheral blood mononucleated cells (PBMCs) were separated using the density gradient method by Histopaque® 1077 (Sigma-Aldrich Company Ltd., Gillingham, United Kingdom).Three milliliters of heparinized blood were mixed 1:1 in RPMI-1640 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 (supplemented with L-glutamine and 25 mM HEPES; Gibco, Grand Island, New York, USA), gentled deposited onto 3 mL of Histopaque-1077 and centrifuged at 400 ×g for 30 min at room temperature.After aspirating the upper layer, the opaque interface containing PBMCs was collected into a clean 15 mL conical tube (Corning Inc., New York, USA).
Cells were washed using 10 mL of RPMI-1640 medium, after centrifuging at 700 ×g for 15 min, the supernatant was discarded and cells were resuspended on 2 mL of RPMI-1640.Using a dilution of 1:10 of this cell solution, concentration was calculated by Neubauer chamber (Brand, Wertheim, Germany).PBMCs were diluted considering the maximum cell concentration to achieve a monocellular layer of cells on a 35 mm Petri dish that is 6.13•10 6 cell•mL -1 .In all irradiation conditions 2 mL of cell solution was added to Petri dish.

Irradiation Conditions
Irradiation assays were done on 35 mm polystyrene Petri dishes, uncoated and tissue culture treated (Nest, Jiangsu, China) (Figure 1-C).After the addition of the PBMCs dilution, the dishes were left undisturbed for 30 min to allow sedimentation according to the method of Virsik et al. [50].The monolayers of cells settled on the bottom of the dishes were exposed separately to 5, 10, 20, and 40 pulses of X-ray emitted from the PF-2kJ device (FWHM ~ 90 ns, dose rate ~ 10 7 Gy•s -1 ), two independent experiments were performed for each irradiation conditions.Additionally, an unirradiated monolayer sample was prepared to evaluate the effect of sham irradiated conditions.An array of 21 TLD-100 dosimeters in the same type of Petri dish was positioned at the anode top, to measure the doses under the same irradiation conditions.

Lymphocyte Culture and Chromosome Analysis
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 After irradiation, cells were collected in a 15 mL conical tube (Corning), washed with 2 mL of RPMI 1640 medium, and centrifuged 10 min to 700 ×g.The supernatant was discarded and cells were resuspended on 0.5 mL of RPMI 1640 medium.The cells were incubated at 37 °C for 2 h with 4 mL RPMI 1640 medium, supplemented with 18 % of Fetal Bovine Serum (Gibco, Grand Island, New York, USA).To stimulate lymphocyte growth Phytohemagglutinin M (Gibco, Grand Island, New York, USA) 1.8 % v/v was added.Lymphocyte cultures were done in presence of 5-Bromo-2´-deoxyuridine (0.9 % v/v) (Calbiochem, San Diego, California, USA).Since an increase in radiation dose can delay the progression of the cell cycle [51-53], and in order to obtain a sufficient number of cells for analysis, the length of culture were 48 h, 50h and 72h for 5-10, 20 and 40 pulses respectively.In all the cases, Colcemid (Gibco, Grand Island, New York, USA) was added after 45 h of culture to obtain a final concentration of 0.1 μg•mL -1 of Colcemid.
All cultures were incubated at 37 °C.After incubation, cultures were centrifuged for 10 min 700 g and the supernatant was replaced by hypotonic solution (KCl 0.075 M, Gibco, Grand Island, New York, USA) prewarmed at 37 °C.After 10 minutes of treatment with the hypotonic solution at 37 °C cultures were centrifuged 5 min 700 ×g, and cells were fixed with methanol and acetic acid (3:1).For the cytogenetic analysis 2 to 3 days old slides were stained by the fluorescence plus Giemsa technique, using Hoechst 33258 stain in pH 6.8 phosphate buffer.
Chromosome analysis was carried out exclusively on first division metaphases containing 46 centromeres.The dicentric chromosomes (dic), tricentric chromosomes (tric), and centric rings (r) were only considered when their corresponding acentric fragment was present.Acentric fragments not related to a multicentric or a ring chromosome were recorded as extra acentric fragments (ace).For each irradiation condition, and following international criteria in cytogenetics dosimetry [34], the number of metaphases analyzed 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 was approximately 500, or those needed to score 100 chromosome aberrations.All metaphases with chromosome aberrations were analyzed independently by two scorers.
During the analysis, the mitotic index was also calculated [34].

Statistical Analysis
To check if the distribution of chromosome aberrations after each irradiation condition followed a Poisson distribution, the dispersion index, variance/mean, and the normalized unit of this index, the U-test, were used [54].Differences among irradiation conditions were evaluated by a one-way ANOVA test.Ordinary least squares method was used to calculate the linear regression between the dose and the frequency of chromosome aberrations.

Results
The average dose (±SD) of TLD measurements at 5, 10, 20 and 40 pulses are 0.12 (±0.02)Gy, 0.14 (±0.03)Gy, 0.22 (±0.06)Gy and 0.47 (±0.09)Gy respectively.From 5 to 10 pulses there was a slight non-significant increase in the mean dose, but from 10 to 40 pulses there was a linear increase in the mean dose (Figure 2).The slope of the linear regression indicates a mean of 12 mGy per pulse.
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.Figure 3 shows microphotographs of representative metaphase for different X-rays pulsed conditions.Cytogenetic results are summarized in Table I.Eight out of 10 total irradiated samples could be analyzed to achieve ~ 500 cells or 100 dicentrics plus rings (40 pulses irradiation condition), and only in two cases, the number of cells analyzed was between 400 and 450 (at 5 and 20 pulses).The mitotic index was always higher than 2.5 %, which is considered a reference value in radiation cytogenetics [34].In this work, the mitotic index seems not to be influenced by the number of X-rays pulses.One-way analysis of variance (ANOVA) is used to determine statistical significance differences between the chromosomal aberrations scoring on different exposure conditions, for dicentrics and extra acentric fragments, there was a clear increase as the number of pulses increased (p< 0.0001 in both cases).Significant differences were also observed for tricentric chromosomes obtained after 20 and 40 X-rays pulses (p<0.0001), and centric rings obtained for 5, 20, and 40 X-rays pulses (p<0.0002).The frequency of centric rings show a tendency to increase with the number of pulses.When total chromosome aberrations 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

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were considered, there was a clear increase (p < 0.001).When the replicas were compared, similar frequencies of CA were observed.These results show the irradiation methodology and experimental setup of PF2kJ are robust.The chromosome aberrations frequency was calculated as the ratio of dicentrics plus rings and the total cells scored, Table II shows these results and dicentric plus ring cell distribution.After uniform exposure to low-LET radiation, dicentric cell distribution follows a Poisson distribution where the ratio between the variance and the mean, the dispersion index (DI) tends to 1, the DI is higher than 1 in all cases, indicating a tendency to show overdispersed values.This overdispersion was significant (U-test > 1.96) in one replica of each irradiation condition.

Discussion
The dicentric chromosomes and centric rings are two different kinds of unstable chromosome aberrations, they are specific to ionizing radiation with a clear dependence on dose, dose-rate and radiation quality.To the best of our knowledge, this work is the first study where cytogenetic biomarkers are analyzed in order to evaluate the biological effect of X-rays pulses at low doses (<0.5 Gy), ultra-high dose rate (107 Gy•s-1) and low energies (8-10 keV).
A monolayer of peripheral mononucleated cells (PBMCs) settled on the bottom of a Petri dish was used, in order to avoid a depth dose gradient in the irradiated sample due to the strong attenuation of such low photon energies.Additionally, the irradiation of unstimulated lymphocytes allows us to have all cells in the same phase of the cell cycle, 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 in this case in the G0 quiescent stage.According to the irradiation conditions presented in [50,[55][56][57][58], to evaluate the induction of chromosome aberrations by conventional X-rays of low energies, some important factors have to be taking into account: i) a monolayer of cells, ii) cellular cycle control and iii) temperature control on irradiation assays.In the present study, petri dishes were kept at 37 °C before and just after irradiation, and although irradiations were not performed at 37 °C, they were above 17 °C as it is suggested by Gumrich et al. [59].
Previous studies have shown that higher doses of radiation result in an increased frequency of chromosome aberrations and a delay in cell cycle [51][52][53].Consequently, the length of lymphocyte culture was set between 48-72 hours, depending on the number of pulses.All mitotic indexes were above 5 %, indicating that culture results were satisfactory.Because the cytogenetic analysis was restricted to first division metaphase, underestimation due to the analysis of second or third division metaphases was avoided.
Radiation exposures at ultra-high dose rates (10 6 -10 7 Gy•s -1 , UHDRs in the present study.As it can be seen, the observed frequencies are higher than all the frequencies that were estimated for the above indicated curves.A weighted least-squares approximation was used to fit the data for each reference radiation quality.Considering irradiation conditions were performed at low doses values (< 0.5 Gy), and in order to compare the biological effect of X-rays pulses with 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 fitting for reference radiations quality (α particles, X-rays and γ radiation).RBEM values are shown in Table III, these results indicate that the photon energy of pulsed X-rays emitted by PF-2kJ is more effective compared even with high LET radiation.In this study, the results show effects as those that are expected after high LET radiation exposures, indicating a different behavior of pulses radiation compared to conventional radiation.The intercellular distribution of dic+r showed a significant overdispersion at 5, 10, 20, and 40 pulses, where the u-test were 4.6, 2.75, 3.21, and 3.09 respectively.Since it is well known that for low-LET radiation dicentric and dicentric plus ring cell distribution agrees with the Poisson distribution, where the variance is equal to the mean.
In the present study the overdispersion (variance > media) observed was unexpected.For these chromosome type aberrations overdispersion is expected after non-homogeneous exposure to low-LET radiation, or after high-LET radiation exposure.However, in this work we observed tricentric chromosomes, these multicentric configurations are rarely observed after low doses of low-LET exposure, and common after irradiations to low doses of high-LET [66-67].
Our results suggest that the pulsed X-rays of low-energy in low-dose range, with ultrahigh dose rate interact distinctively with the DNA.Considering PF-2kJ has these characteristics it is necessary to gain a better understanding of mechanisms underlying the DNA damage induced by X-rays pulses such as by analyzing the formation of complex chromosome aberrations using fluorescence in situ hybridization (FISH) techniques, or evaluating the repair kinetics of the double strain break by analyzing the γ-H2AX foci [68].The incorporation of others cytogenetic biomarkers could contribute to the characterization, since they allow direct evidence of the effects of radiations in biological systems.

Conclusions
In the present study our results evidence a different radiobiological response of PBMCs to pulsed irradiation.The presence of tricentrics and the overdispersion observed at low 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 doses, ultra-high dose rate and low energies of X-rays emitted by a PF-2kJ, suggest that the biological effect of X-rays pulses seems to be like high LET.The RBEM analysis confirms this observation, where the pulses emitted by PF-2kJ are more effective inducing CA compared even with high LET radiation.
Among other effects induced by ionizing radiation (IR), like X-rays, the double-strand breaks in the DNA molecule are the most important.Consequences of these lesions are mutations and chromosome aberrations due to unrepair or misrepair during the cellular cycle division[31].Dicentric chromosomes are formed by the misjoining of two broken chromosomes that carry the centromeric region of each chromosome; the formation of centric rings results from the erroneous joining of a chromosome that breaks into two arms, joining itself.In either of these cases the remaining acentric chromosome fragments also join, forming what is known as an acentric fragment[32].These chromosome aberrations (CA) are almost exclusively induced by IR[33].The CA frequency is also commonly used as a cytogenetic biomarker of dose exposure[34].The analysis of this biomarker in peripheral blood lymphocytes (PBL) is considered a robust and "gold standard" biological dosimetry method and an important tool in the area of radiation protection[35][36].Dicentric chromosomes and centric rings are the most reliable and repeatable method for comparing biological response for a wide range of doses and qualities of ionizing radiation[37].

Figure 4 Figure 4 .
Figure4shows the frequencies of different chromosome aberrations, calculated from the ) have been shown to manifest differential radiobiological responses, and induced less damage, compared to conventional (CONV) dose rates (0.001-0.4Gy•s -1 )[5].The increase in radical-radical recombination and oxygen depletion are the main hypotheses to explain a reduced yield of biological lesions at ultra-high dose rates[60].Previous cytogenetic studies using UHDR have shown a decrease in the induction of chromosome aberrations when the number of pulses or the dose rate increased[61][62][63].It should be noted that these earlier studies were performed in a higher energy range (in the order of MeV) and at higher radiation doses (2-8 Gy).In contrast, in the present study, we observed an increase in chromosome aberrations with the number of pulses.Our results seem to suggest that at low doses and for low-energy X-rays delivered at UHDR, there is a major biological effectiveness in producing DNA damage.Acharya et al. [63]  observed an increase of 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: 10.1063/5.0141529micronuclei yield when doses were delivered by multiple pulses compared with those delivered by single pulse, especially at higher doses of 50 and 25 ns pulses.This seems to indicate that for fractionation strategy short time of X-rays pulses (90 ns) which are obtained from PF-2kJ, could be an important parameter on chromosome aberrations induction.Four reference dose-effect curves were applied to estimate the chromosome aberration frequency expected at the physical doses reported in the present work (TLD-100): a αparticle curve of 241 Am 2.7 MeV, 0.1 Gy•min -1 [55]; two X-rays curves, one of 180 kVp, 0.27 Gy•min -1 [64], and another of 10 keV, 0.5 Gy•min -1 [42]; and one γ radiation curve of 60 Co, mean energy 1.25 MeV, 1.2-1.1 Gy•min -1 [65].The frequencies values estimated are shown in Figure 5, and compared with the frequency of dicentrics plus rings observed

Figure 5 .
Figure 5.Comparison of dicentric plus rings (dic+r) frequency observed in the present study (red circles), and frequencies calculated using previously published curves for α- 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

Table I
Cytogenetic results obtained from lymphocytes irradiated with X-rays pulses.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.

Table II .
Intercellular distribution of dicentric chromosomes plus centric rings after X-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.

Table III .
Linear regression fit coefficient, and the biological relative effectivenessThis 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.