18F-FDG-induced DNA damage, chromosomal aberrations, and toxicity in V79 lung fibroblast cells

Journal Pre-proof 18 F-FDG-induced

DNA damage, chromosomal aberrations, and toxicity in V79 lung fibroblast cells Tanmoy Mondal, Amit Nautiyal, Milee Agrawal, Deepanjan Mitra, Alpana Goel, Subrata Kumar Dey

PII:

S1383-5718(19)30187-1

DOI:

https://doi.org/10.1016/j.mrgentox.2019.503105

Reference:

MUTGEN 503105

To appear in: Mutagenesis

Mutation Research - Genetic Toxicology and Environmental

Received Date:

13 June 2019

Revised Date:

4 October 2019

Accepted Date:

5 October 2019

Please cite this article as: Mondal T, Nautiyal A, Agrawal M, Mitra D, Goel A, Kumar Dey S, DNA damage, chromosomal aberrations, and toxicity in V79 lung fibroblast cells, Mutation Research - Genetic Toxicology and Environmental Mutagenesis (2019), doi: https://doi.org/10.1016/j.mrgentox.2019.503105

18 F-FDG-induced

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F-FDG-induced DNA damage, chromosomal aberrations, and toxicity in V79 lung

fibroblast cells Tanmoy Mondal1, Amit Nautiyal2, Milee Agrawal1, Deepanjan Mitra2, Alpana Goel3, Subrata Kumar Dey1,* [email protected] 1

*Department of Biotechnology, Maulana Abul Kalam Azad University of Technology,

BF-142, Sector-I, Salt Lake, Kolkata-700 064, West Bengal, India. Institute of Nuclear Medicine & Molecular Imaging, Advance Medicare & Research Institute,

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P-4&5, Gariahat Road Block-A, Scheme-L11, Dhakuria, Kolkata-700029, West Bengal, India. Amity Institute of Nuclear Science & Technology, Amity University, Noida, Delhi, India.

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Address for correspondence to: Subrata Kumar Dey, Department of Biotechnology, Maulana Abul Kalam

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Azad University of Technology, BF-142, Sector-I, Salt Lake, Kolkata-700 064, West Bengal, India. Highlights

Effects of 18F-FDG 511 keV gamma rays were studied in V79 cells in vitro.

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DNA damage, cell cycle, and toxic effects were measured.

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γH2AX and JC1 endpoints showed significant responses to radiation.

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Both chromosome and chromatid types of aberrations were observed and aneuploidy increased.

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Abstract

F-FDG PET/CT imaging is used in the diagnosis of diseases, including cancers. The principal photons

used for imaging are 511 keV gamma photons resulting from positron annihilation. The absorbed dose varies among body organs, depending on administered radioactivity and biological clearance. We have attempted to

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evaluate DNA double-strand breaks (DSB) and toxicity induced in V79 lung fibroblast cells in vitro by 18FFDG, at doses which might result from PET procedures. Cells were irradiated by

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F-FDG at doses (14.51

and 26.86 mGy), comparable to absorbed doses received by critical organs during PET procedures. The biological endpoints measured were formation of γ-H2AX foci, mitochondrial stress, chromosomal aberrations, and cell cycle perturbation. Irradiation induced DSB (γH2AX assay), mitochondrial depolarization, and both chromosome and chromatid types of aberrations. At higher radiation doses,

increased aneuploidy and reduced mitotic activity were also seen. Thus, significant biological effects were observed at the doses delivered by the 18F-FDG exposure and the effects increased with dose. Keywords: PET-CT, low-dose radiation, DNA strand breaks. 1. Introduction PET (Positron Emission Tomography) provides quantitative and qualitative information on the distribution of PET radiopharmaceuticals in the body. PET integrated with computed tomography (CT) has emerged as a powerful imaging tool. 18F-FDG (18F-fluorodeoxyglucose) is a radiopharmaceutical which emits

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positively-charged beta particles (positrons) [1]. When a positron interacts with an electron, an annihilation

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process takes place, where the masses of the positron and the electron are converted into energy and two 511 keV photons are emitted at 180° to each other [2]. These photons are the major source of the radiation dose

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received by the patient’s body. The total effective dose received by a patient from a combined PET/CT procedure is about 15 mSv, equivalent to several years of background radiation or as many as 765 ordinary X-

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rays [3, 4]. PET/CT has proved to be a very sensitive dual-modality imaging method for detection of cancer,

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improving treatment and outcomes [4, 5]. However, the radiation dose received by the patient is of concern. A standard PET/CT procedure involves an intravenous injection of 10 mCi (350 MBq) 18F-FDG. The resulting

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absorbed doses to organs depends on their metabolic activity, e.g., brain, 10-36 mGy; heart, 16-51 mGy; kidneys, 7-23 mGy [2]. Many studies have reported the frequencies of chromosomal aberrations and estimated the probability of cancer induction, due to moderate to high doses of ionizing radiation [6, 7]. However, biological effects and health risks may also result at doses <100 mGy. High radiosensitivity has been observed

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in cancer patients without any obvious immunodeficiency, resulting in severe radiation therapy side effects [8, 9]. Some published data have described

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F-FDG induced DNA damage and the relative biological

effectiveness in a mouse model [2, 10] and a few studies have shown radiation-induced DNA damage in patients after CT and interventional imaging procedures [4, 11, 12,] but little data is available with respect to PET radiopharmaceuticals. Evaluation of the biological effects of low-dose ionising radiation is needed for optimal management of radiological investigations.

Ionizing radiation causes molecular damage, altering the primary structure of DNA [13]. Double-strand breaks (DSB) are the most critical lesion [22]. The γ-H2AX foci formation method is a very sensitive technique for detection of DSB. Low-dose exposure from CT procedures can induce DSB [14]. Unrepaired DSB may lead to chromosomal abnormalities, depending on damage amount and repair [15]. Thus, the assessment of chromosomal abnormalities can be a useful tool in the estimation of biological effects of ionizing radiation. Altered DNA content is strongly correlated with cellular stress [16]. Flow cytometric analysis of DNA and cell proliferation methods have become useful techniques in which cell numbers can be analyzed

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expeditiously. Our previous work [17] demonstrated DNA damage and cell apoptosis at 8.22 mGy, the average

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dose received by tissues during a PET scan.

We have evaluated DSB induction and toxicity of 18F-FDG at a radiation dose which may be absorbed

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by critical organs during a PET procedure. Formation of γ-H2AX foci, mitochondrial stress, chromosomal aberration induction, and cell cycle changes were analysed in V79 lung fibroblast cells.

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2. Materials and methods

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2.1. Cell culture and irradiation

The Chinese hamster lung fibroblast V79 cell line was obtained from ATCC (US) and maintained as

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monolayers in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco), and 100 U penicillin + 100 µg streptomycin/ml (Gibco). Cells were cultured in humidified atmosphere with 5% CO2 at 37°C in 75 cm2 flasks (Corning, NY). Irradiation was done at the Department of Nuclear Medicine & PET/CT, Advance Medicare & Research Institute (AMRI), Kolkata.

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Cells were first seeded at a density of 106 cells/flask and then irradiated by 18F-FDG in cold buffer condition using 14.51 or 26.86 mGy absorbed doses. An untreated control was kept during all experimental procedures. 2.2. Dose calculation

The total absorbed dose was calculated according to our previous work by Mondal et al. [17]. Here, the cells were exposed using a 37 MBq source of 18F-FDG for 15 or 30 min to achieve the desired dose. Dose selection was based on the doses received by organs during the WB PET/CT procedure. For dose estimation,

the decayed radioactivity was estimated for each minute until 30 min using the formula [18] - 𝑁 = 𝑁0 𝑒 −𝜆𝑡 (1) where N0 is the number of undecayed nuclei at time t and λ is the decay constant. We converted radioactivity into dose rate (mGy/h) [19]. Radioactivity was first converted into exposure rate, using the specific γ-ray constant of

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F, 5.65 R-cm/mCi-h. The estimated doses for each minute were

summed to give the cumulative doses for 15 min or 30 min, 14.51 or 26.86 mGy, respectively. To estimate the dose rate, suitable correction factors were applied related to attenuation and distance. Attenuation by the medium [18, 20] (1 cm including 2 mm plastic) was corrected using linear attenuation coefficient (μ) and

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thickness(x). 𝐼 = 𝐼0 𝑒 −𝜇𝑋 (2) where I is the dose after transmission from distance X, I 0 is the initial dose, e is a

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proportionality constant that reflects the total probability of a dose being scattered or absorbed, μ is the linear attenuation coefficient, and X is distance.

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2.3. γ-H2AX foci assay

Immediately after irradiation, samples were placed on ice. Before γ-H2AX foci analysis, all samples

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were incubated at 37°C, 5% CO2 for 30 min to allow phosphorylation of H2AX histones. Irradiated cells were

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then fixed in 4% paraformaldehyde (pH 7.4) for 15 min and washed three times in PBS (5 min each). Fixed cells were then permeabilized on ice using 0.1% Triton X-100 (15 min) and again washed gently with PBS.

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Next, cells were blocked in 10% FBS and 0.1% Tween 20 in PBS for 30 min and further incubated with mouse anti-H2AX phosphorylated monoclonal antibody (ser 139) clone (Millipore, UK) in the dark for 2 h at room temperature. Washing of slides was done with 0.1% Tween 20 in PBS and cells were again incubated with Alexa Fluor 488 goat anti-mouse IgG secondary antibody (1:500) (Invitrogen, Germany) for 1 h at room

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temperature. Cells were again washed four times in PBS for 5 min and Vectashield mounting medium with 4, 6-diamidino-2-phenylindole used for imaging. Images were obtained by using fluorescence microscope (Leica DM300, Leica Microsystems, Wetzlar, Germany). Foci analysis was done with imageJ software [21]. During the counting process, the Cell Counter plugin was used as an extension with the imageJ software and at least 200 cells were counted for determination of frequency, average number, and average size of foci [17]. 2.4. JC-1 Fluorescence imaging

The

JC-1

(5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazoylcarbocyanine

iodide)

mitochondrial

membrane potential test was used to test mitochondrial behaviour. JC-1 is a fluorescent indicator; small changes in ΔΨM are marked by green or red fluorescence shifts and can be evaluated by fluorescence microscopy. The protocol of Zhang et al. [22] was followed. Irradiated samples were centrifuged at 1700 rpm for 10 min and the pellet was mixed gently with JC-1 staining solution (100 µl per ml medium). Samples were incubated in a CO2 incubator at 37°C for 15-30 min and centrifuged again. The collected pellet was mixed with 300 µl assay buffer (Cayman kit 10009172) and analysed by fluorescence microscope and flow cytometry

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(BD FACSVerse TM, BD Biosciences USA) with a 488 nm argon laser light source.

Cells were cultured at 106 cells/flask and irradiated by

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2.5. Chromosome aberration study

F-FDG. Exponentially growing V79 cells

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have a doubling time of 12-14 h. In our study, irradiated cells were harvested at 4 h incubation to obtain late G2/S phase [23]. Colcemid (5 mg/ml) was added to the culture medium 2 h before harvesting. After

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incubation period, cells were harvested and treated with hypotonic 0.56% KCl solution for 15 min and then

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fixed in freshly prepared acetic acid: methanol (1:3). Changing of fixative was done at least three times before the cell suspension was dropped onto a chilled pre-cleaned microscopic glass slide and dried at room

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temperature for at least two days [24]. Giemsa staining was applied for scoring metaphase chromosomes. at least 300 metaphase cells were analysed for scoring number of aberrations, under a binocular light microscope (Carl Zeiss, Germany) and classified according to Savage’s method [25]. Cytotoxicity analysis was done by calculating the mitotic index (MI) percentage, counting the number of metaphase cells present in 1000 cells

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per sample.

2.6. Cell cycle and DI analysis Cell cycle phase distribution of nuclear DNA was checked. V79 cells, 1 × 106, were fixed with p-

formaldehyde, 3%, and permeabilized with Triton X-100, 0.5%. Nuclear DNA was labelled with propidium iodide (PI, 125 µg/mL) after RNase treatment using Cycle TEST PLUS DNA reagent kit (BD Biosciences340242) [26]. A flow cytometer (BD FACSVerse TM, BD Biosciences US) with a 488 nm argon laser light source was used for checking the cell-cycle distribution.

Flow cytometric DNA Index (DI) analysis was performed as described above. Here also, nuclei were stained with PI and DNA histograms were generated using FACS. Untreated V79 cells served as an internal control for normal (diploid) DNA content. The staining procedure for DNA ploidy analysis requires a test sample of 5 × 105 cells and we prepared an additional sample tube of the irradiated sample mixed or “spiked” with an unirradiated sample at 2:1 ratio. Histograms having a G0/G1 peak distinctly separate from diploid cells (confirmed by the addition of normal control cells) were considered to have aneuploid DNA content. The Dl was calculated using the ratio of the modal fluorescence channel numbers of the aneuploid and diploid (G0/G1)

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peaks. Cells having the ratio of G0/Gl peak is 1.0 (DI = 1.0) were considered diploid.

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2.7. Statistical analysis

All statistical analysis was calculated using Student’s t-test. Reported data are represented as means

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±SEM for three independent experiments. GraphPad Prism (Version 7) was used to analyse all data. To study the differences among three or more means, we used one-way analysis of variance (ANOVA). We considered

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differences statistically significant at p < 0.05 (*p<0.05, **p<0.01, ***p<0.001).

3.1. γ-H2AX foci induction.

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3. Results

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The γH2AX assay is a reliable and sensitive biomarker for detection of DNA DSBs. We evaluated the number, size and the specific rate of foci formation in V79 cells. Initially, in the control sample, we found few foci, 0.22 ±0.15 per cell, but the number was higher in irradiated samples (Fig. 1B). At 14.51 and 26.86 mGy, the numbers of foci observed were 1.8 ±0.25 and 2.81 ±0.14, respectively. The average sizes of the foci were

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0.03 ±0.02, 1.78 ±1.02, 3.64 ±1.95 for controls, 14.51 mGy and 26.86 mGy, respectively (Fig. 1C). The rate of appearance of foci also increased at the higher dose (Fig. 1A & 2). 3.2. Estimation of mitochondrial depolarization by JC-1 staining. Loss of mitochondrial transmembrane potential (ΔΨM) is linked to mitochondrial dysfunction and cellular damage. Mitochondria supply energy required during differentiation. JC-1 is a lipophilic cationic probe which differentiates healthy cells by using potential (ΔΨM) dependent J-aggregate formation (green or red fluorescence) [27]. To determine whether low dose radiation may change mitochondrial dynamics,

irradiated V79 cells were subjected to fluorescence staining; microscopy shows a significant change in the appearance of JC-1 monomers at the higher dose (Fig. 3A, 3B, 3C). Flow-cytometric analysis (Fig. 3a, 3b, 3c) of JC-1 stain shows a significant increase in depolarization with increasing dose, 20.03, 56.17, and 70.23 for control, 14.51 mGy, and 26.86 mGy, respectively (Fig. 4).

3.3. Chromosome aberration study V79 cells contain 22 chromosomes. A detailed metaphase spread plate and a karyotype image of

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V79 chromosomes are given in Fig. 5 A, B. Irradiated samples showed the following types of aberration: chromatid gaps, chromatid breaks, chromatid deletions, terminal deletions and some interstitial deletions,

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complex rearrangements, chromosome gaps, chromosome breaks, dicentrics and rings. An example of

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chromosome aberrations in a metaphase spread of γ-irradiated V79 cells is shown in Fig. 6 and the numbers of aberrant chromosomes are shown in Table 1.

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3.4. Flowcytometric analysis of cell cycle phase distribution and nuclear DNA index

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Fig. 7 shows the cell cycle distribution after irradiation. Exponentially growing V79 cultures contained about 52% G0/G1, 12% S-phase, and 13% G2+M phase cells. A decrease in The G0/G1 percentage increased following irradiation group, to 67% and 80% at 14.51 and 26.86 mGy, respectively, whereas the proliferative

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phases S and G2/M combined decreased (13% control, 10% at 14.51 mGy, 8% at 26.86 mGy). Fig. 8 shows a distinct G0/G1 peak, separated from diploid cells (confirmed by the addition of normal control cells), considered to have aneuploid DNA content. Both irradiated groups were found to have nearly diploid DNA

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indices, 1.12 and 1.32. 4. Discussion. 18

F-FDG is widely used for PET imaging. The half-life of fluorine-18 (positron (β+) emission) is 109.7

minutes. A typical clinical protocol involves administration of 350–750 MBq 18F-FDG and the residence time in internal organs is 2.38 ± 0.12 h [2]. Several dose estimates from the internal administration of 18F-FDG in humans have been reported, most of them based on bio-distribution studies. Organs having higher metabolic energy requirements receive higher radiation doses: brain, heart, and the excretory system, including kidneys

and bladder [28]. An important aspect of this study is evaluation of the potential risks of radiation exposure of internal organs. Such risks must be understood so that risk-benefit ratios can be assessed. In the present invitro study, we delivered 14.51 or 26.86 mGy absorbed doses to a cell line. The absorbed dose received by the cells are relatively high and equivalent to the absorbed dose received by active metabolic organs and tissues during the PET procedure. V79 cells were used as an in vitro model. Even a single unrepaired DSB can cause chromosomal aberration, apoptosis, or mutation [29, 30]. Following DSB production, H2AX molecules are rapidly phosphorylated; these phosphorylated histones can

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be detected by the γH2AX assay [27]. γH2AX foci increased at 26.86 mGy absorbed dose compared to controls

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(p-value <0.0001). Huang et al. [31] showed that apoptotic cell death is directly linked with increased levels of γH2AX phosphorylation. To maintain the function of the respiratory chain, maintenance of mitochondrial

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membrane potential (ΔΨm) is necessary; the loss of ΔΨm leads to cell death [32]. Irradiated samples showed low ΔΨm, confirmed by FACS analysis, where the percentage of depolarized cells significantly increased.

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DNA DSB are lesions leading to chromosomal aberrations [33] and ionizing radiation induces

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chromosome abnormalities. Such abnormalities increased in a radiation-dose-dependent manner. A few studies have already reported chromosomal abnormalities following computed tomography scan procedures [4, 34]

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but the relationship between radiation dose at low doses (<100 mSv) and induction of aberrant chromosomes is unclear. In this study, both chromosome and chromatid types of aberrations were observed; and break, dicentric and ring type of chromosomes were present predominantly. Such aberrations are known to be lethal and not passed on to progeny cells. The percentage of dicentric chromosome formation also increased

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significantly after irradiation and this is only possible when radiation exposure causes DNA strand breaks. Inter-chromatid exchange-type aberrations (Fig. 6) were also seen. The exchange forms when interacting lesions occur in the arms of different (homologous or non-homologous) chromosomes [25] and this may be due to DNA DSB. Since γ-radiation induces DNA damage, cellular stress and abnormal chromosomes, we used flow cytometry analysis to test whether γ-radiation affected cell-cycle progression. We observed increased G0/G1 cells and decreased S and G2/ cells following irradiation (Fig. 7). These results indicate that the entries of cells

into S-phase and into the primary mitosis phase were delayed by irradiation and caused a severe reduction in mitotic movement. Under normal conditions, cells at G0/G1 phase synthesize mRNA and proteins required for DNA synthesis as well as proteins such as cyclins and cyclin-dependent kinases (cdk) that regulate cell-cycle progression [35]. Damage-sensing proteins, e.g. kinases, ATM, and ATR, are associated with maintenance of genome integrity. DNA damage may lead to inhibition of the cell cycle, mediated by a complex network of cell signalling pathways [36, 37]. One may assume that radiation-induced damage is responsible for the observed arrest at G0/G1. Fournier and Taucher-Scholz [37] reported that radiation causes G1 arrest during

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fibroblast cell division. A similar result is also observed by DNA index analysis. A DNA content ratio between

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sample and control groups of 1, indicates a diploid chromosome number [38]. In our study, both irradiated groups had close to diploid DNA indices, implying that aneuploidy increased after gamma radiation,

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presumably due to the effects of DNA damage. In conclusion, our in vitro study tested the effects of 18

F-FDG irradiation at doses similar to the

F-FDG exposure during PET scan procedures. The results indicate that

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absorbed doses due to

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18

F-FDG

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induces DNA DSB, chromosomal abnormalities, and cytotoxicity. Funding source

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This work was supported by the Council of Scientific & Industrial Research (CSIR), India [File No. 09/1213(0001)/2018-EMR-1] Conflicts of Interests

The authors declare no conflict of interest.

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Acknowledgement

We thank Dr Sandip Pal, Department of Zoology, Barrackpore Rastraguru Surendranath College, Barrackpore, West Bengal, Priyanka Ghosh of Department of Biotechnology, Maulana Abul Kalam Azad University of Technology, Kolkata, West Bengal and Dr Aruna Kaushik, Department of Cyclotron & Radiopharmaceutical Sciences, Institute of Nuclear Medicine & Allied Sciences, Delhi, India for wholehearted cooperation throughout the work.

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T. Ishida, Y. Nakano, Y. Kihara, C.N. Hayes, H. Aikata, K. Chayama, T. Ito, K. Awai, S. Tashiro, Chromosomal abnormalities in human lymphocytes after computed tomography scan procedure,

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Radiat. Res. 190 (2018) 424-432.

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37. C. Fournier, G. Taucher-Scholz, Radiation induced cell cycle arrest: an overview of specific effects following high-LET exposure, Radiother. Oncol. 73 (2004) S119-22.

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38. Z. Darzynkiewicz, H.D. Halicka, H. Zhao, Analysis of cellular DNA content by flow and laser scanning cytometry, Adv. Exp. Med. Biol. 676 (2010) 137–147.

Figure 1. Measurement of γ-H2AX: A. γ-H2AX foci formation, after irradiation with 18F-FDG. γ-H2AX foci are red and nuclei were stained with DAPI (blue). B. Histogram of mean foci/cell scored from three

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independent experiments (control and irradiated samples). C. Histogram of mean focus size (control and irradiated samples). (**p < 0.001, ***p < 0.0001 by Student’s t-test; value obtained from three independent experiments).

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Figure 2.Distribution of γ-H2AX foci in V79 cells for control, 14.51, and 26.86 mGy samples.

Figure 3. Measurement of mitochondrial membrane potential: Upper three images: V79 cells with an accumulation of JC1 stain in the mitochondria (in red): (A) non-irradiated V79 cells (control); cells irradiated

at 14.51 mGy (B) and 26.86 mGy (C). Irradiated samples appears in green because JC1 aggregates remain in the cytosol. Lower three panels are flow cytometric analysis of control and treated samples; control (a), 14.51

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mGy (b), 26.86 mGy (c) irradiated samples.

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Figure 4. Comparison of accrued depolarised cell percentage by flow cytometric method (*p < 0.01, ***p <

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0.0001 by Student’s t-test; three independent experiments).

Figure 5. Karyotype analysis A. Giemsa-stained metaphase chromosome plate of non-irradiated V79 cell. B. Karyotype of V79 chromosomes in an 11 pair presentation.

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Figure 6. Chromosomal aberrations: Giemsa stained metaphase chromosome plates showing an example of diverse aberrant chromosomes- chromatid breaks (a), dicentric formation (b), inter-exchange or translocation

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(c, e, f), terminal break (d), ring (g).

Figure 7. Flow cytometric analysis of cell cycle distribution: Unirradiated control (top), 14.51 mGy (middle), and 26.86 mGy (bottom) irradiated samples. Nuclear DNA was determined by flow cytometry using propidium iodide (PI) as DNA-binding fluorochrome. Histogram display of DNA content (X-axis, PIfluorescence) versus counts (Y-axis) is shown.

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Figure 8. DNA index (DI) analysis: Both panels represent G0/G1 phase of cell cycle for each sample. Cells in both panels were irradiated with 14.51 and 26.86 mGy respectively and spiked with unirradiated control cells at 2:1 ratio. The DI was calculated using the ratio of G 0/Gl peak. DNA content (X-axis, PI-fluorescence)

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versus counts (Y-axis) is shown.

Table 1. Distribution of the different types of chromosomal aberrations in 300 cells analysed per treatment and mitotic index (MI) observed at irradiated and unirradiated groups. Aberrations Samples

MI (%)

Chromatid-type

Chromosome-type

gap

breaks

exch

breaks

dic

ring Total

Control

15.1

0

2

1

1

0

0

4

14.51 mGy

13.9

6

17

9

8

11

3

54*

26.86 mGy

11.6

8

22

14

9

13

5

71*

Aberrations type: dic- dicentrics; exch- exchanges.

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*Statistically significant when compared with untreated control (P < 0.001).