The role of intrinsic radiosensitivity in the low-dose adaptive response induction in human peripheral blood mononuclear cells

INTRODUCTION Although high doses of ionizing radiation (HDIR) are now accepted to be detrimental for living organisms exposed to these types of radiation, there are still uncertainties on the biological health effects of low doses of ionizing radiation (LDIR). Radio-adaptive response (RAR) is a phenomenon related to the low-dose range of ionizing radiation (IR), which is defined as being less sensitive to the harmful effects of a high-dose radiation by being exposed to a prior conditioning dose.[1] Because of the existed experimental evidence, adaptive response (AR) induction is not a constant phenomenon among all individuals.[2-5] Furthermore, there are several factors such as priming and challenging dose, the time interval, and the dose rate that could affect the AR results. Furthermore, inter-individual genetic differences including intrinsic radiosensitivity could be considered as one of the other reasons associated with AR induction dissimilarity.[6] Intrinsic radiosensitivity, which is one of the radiobiological concepts causing different cell’s and tissue’s reactions among different populations, can be described as the susceptibility of an individual to IR and is also associated with some genetic material alternations.[7] As mentioned above, the concept of individual radiosensitivity depending on individual genetic background has been supported by data from previous studies.[8] In radiotherapy, inherent radiation sensitivity seems to be the most logical reason for the differences among patients’ responses.[9] Radiation-induced complications in patients as a one of the main challenges in radiation therapy affected by high intrinsic radiation sensitivity.[10] Individually assessment of radiosensitivity holds the hope that cancer therapy moves towards personalized medicine.[11] Generally, to have the ability of practically utilizing the protective effects of AR, it is essential to notice its mechanism and related factors. This hypothesis should be investigated whether the incidence of AR is affected by radiosensitivity. One of the recent observations is the contradictory occurrence of adaptive effect in healthy and tumor cells, which proves at the cellular levels in cells with different sensitivity.[12] Therefore, in the present study, the relationship between the AR occurrence and the intrinsic radiosensitivity, as the missing link in this chain, was evaluated. There is significant evidence in the relevant literature cytogenetic AR incidence. Therefore, this method was used to investigate the relationship between AR and radiation sensitivity. In terms of radiation protection, it would be useful if the results obtained from radiosensitivity grading help in identifying people who are the most likely ones to experience AR. MATERIALS AND METHODS Blood sample’s collection Venous blood samples were obtained from eight healthy male nonsmoker donors aged between 25 and 35 years who were included in two different experiments. Afterward, peripheral blood mononuclear cells (PBMCs) were isolated from diluted blood using Ficoll (Cedarlane, Canada) density gradient centrifugation. The separated layer was then washed twice with phosphate-bufferedsaline. Collection of the blood samples was approved by the ethics committee of Mashhad University of Medical Sciences (approval code: IR.MUMS.MEDICAL.REC.1397.654) and written informed consent was obtained from all donors before blood collection. Intrinsic radiosensitivity As radiosensitivity is caused by the adverse tissue reactions related to cell death, it is better to perform the radiation sensitivity assay in terms of all the three major cell death pathways. Thus, clonogenic assays are known as the gold standard for radiosensitivity measurement. To specify intrinsic radiosensitivity using endpoint limiting dilution assay (specific clonogenic cell survival assay for suspension cultures), three aliquots obtained from each blood sample were used to consider technical replication.[13] Subsequently, PBMCs of each blood aliquots were suspended at 2 × 103 cells/ml in RPMI 1640 (Gibco, UK) medium supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, Germany), 1% pen-strep (Sigma-Aldrich, USA), 1% phytohemagglutinin (PHA) (Gibco, USA), 10 IU/ml interleukin 2 (IL-2), 1% inactivated human serum, and 1% nonessential amino acids (Gibco, UK). Lymphocytes were then plated into 96-well U-shaped bottom plates at 10 and 17 μl/well in the control and irradiated groups, respectively. The latter groups were irradiated at a dose of 2Gy and the dose rate of 200 cGy/min with 6 MV X-ray photons at room temperature using plexiglass phantom with 1.5-cm thickness with the Source to Skin Distance (SSD) = 100 cm, the field size = 30 × 30 cm2, and the gantry angle = 180o using a linear accelerator. Afterward, the prepared plates were incubated for 10 days at 37 in 5% CO2 incubator for the purpose of forming colonies by the cells during the incubation time. Colony forming efficiency (CFE) of cells was calculated, as described elsewhere, by scoring the negative wells (containing cells that were unable to form colonies) via an inverted microscope.[13] Surviving fraction at 2 Gy (SF2), as the indicator of radiosensitivity, was measured as the ratio of surviving fractions (–ln (negative wells/scored wells)/plated cells per each well) under the irradiated to the non-irradiated conditions. Chromosomal aberration analysis In the second experiment, lymphocytes suspended in RPMI medium containing 1% pen-strep and 10% FBS were exposed to 2 Gy X-rays by passing 4 h from an adaptive low dose of 0.1 Gy with the dose rate of 50 cGy/min at room temperature. To arrest PBMCs at metaphase, 0.05 μg/ml Colcemid (Gibco, USA) was added 24 h after the PHA stimulation (0.01 ml/ml), 24 h before harvesting. After removing Colcemid by centrifugation, the cells were treated for 10 min with 5 ml KCl hypotonic solution (75 mM) at 37°C. Lymphocytes were then fixed for 10 min in the 5 ml fixative solution (3:1 methanol/acetic acid) at room temperature. Afterward, the slides on which cell suspension was dropped were stained by Giemsa and chromosome aberrations including dicentrics and rings were detected through performing microscopic examination. Statistical analysis To indicate whether AR has significantly occurred, the expected and observed frequencies of chromosome aberrations of the adapted samples were compared using one-sample t-test. Afterward, donors were divided into two groups (AR+, P < 0.05 and AR–, P > 0.05), and mean SF2 values of each group were then evaluated and compared using independent samples t-test. The correlation between SF2 and reduction percentage of chromosome aberrations compared to the expected values was conducted using Pearson’s correlation. Statistical analysis was performed using GraphPad Prism (version 8.3.0) software. RESULTS Individual radiosensitivity To estimate SF2 using a limiting dilution assay, microscopic graphs of wells containing colonies (positive wells) and those with no colony forming (negative wells) were taken and then scored. Results of lymphocytes cloning are shown in Figure 1a, as the average number of negative wells per scored wells ± SD in irradiated and non-irradiated groups for every donor. In addition, lymphocytes’ mean CFE and SF2 values of each donor ± SD are demonstrated in Figure 1c-d.Figure 1: Results of lymphocytes cloning to estimate surviving fraction at 2 Gy (SF2) using the limiting dilution assay (a) the average number of negative wells per scored wells ± SD in irradiated and non-irradiated groups for every donor (b) multiple cell densities were plated to determine the fraction of wells with colony-formation failure. Microscopic pictures of wells containing colonies (yellow wells), wells with no colony forming (white wells), and blank wells (gray) are also shown (c) analysis of clonogenicity to calculate colony forming efficiency and SF2 (d) means value of SF2 ± standard deviation in different donorsAR Chromosome aberrations of each sample were blindly counted, without knowing the results of the radiation sensitivity test. The Chi-square test indicated the homogeneity of the aberration yields between donors (P > 0.05). Furthermore, the expected values for the samples that received both low and high doses of radiations were calculated, as mentioned somewhere else, as follows: low-dose chromosome aberrations + high-dose chromosome aberrations – background chromosome aberrations.[14] These results are presented in Table 1. In addition, a significant incidence of AR was evaluated by the comparison of the expected and observed chromosome aberrations in samples that received a challenging dose pretreated with a priming dose. Table 1.Table 1: Induced chromosome aberrations and AR in different donorsAs it is evident, only four donors have shown AR. Although decreasing frequency of chromosome aberrations in the adapted samples was observed in all of them, AR induction was not statistically significant for the other four donors. Mean SF2 values in AR+ and AR– individuals were found to be significantly (P < 0.05) different as demonstrated in Figure 2b. The results are also indicated a lower mean SF2 (28.03%) through four individuals that have remarkably shown AR compared to the 40.93% mean SF2 in four donors with negative AR manifestation. To find out how the AR and intrinsic radiosensitivity are correlated, the relationship between SF2 values, as the index of radiosensitivity, and the chromosome aberrations reduction percentage in the adapted samples of donors was studied as shown in Figure 2a. Accordingly, as observed, these two factors found to have a significantly inverse correlation (R2 = 0.8097, P = 0.002).Figure 2: (a) Correlation between surviving fraction at 2 Gy (SF2) values and observed chromosome aberrations reduction percentage compared with the expected values in different individuals (b) mean SF2 values in two groups of individuals with different adaptive response incidence. Bars show the standard errors. *Statistically significant (P < 0.05) difference between SF2 valuesDISCUSSION Donors were selected using as close features as possible to eliminate the effect of physiological factors, such as age, gender, and smoking status, on the results of the study. Our findings have approved the variable manifestation of RAR in dissimilar individuals that had been reported earlier.[2-5] Furthermore, the results of the present study assert that the AR induction is highly variable among human beings with different levels of intrinsic radiosensitivity. According to these results, the higher the radiosensitivity, the greater the difference between the expected and observed frequencies of chromosome aberrations. The most radiosensitive donor (%SF2 = 19.7) has exhibited 37.53% chromosome aberrations decrement in the adapted samples, whereas it was about 5.69% in the most radioresistant one (%SF2 = 49.1). This report is in line with the conclusions of two other studies that reviewed the impact of intrinsic radiosensitivity on the AR incidence based on the Ataxia-Telangiectasia Mutated (ATM) nucleoshuttling hypothesis.[15,16] Correspondingly, these reviews stated that the AR phenomenon is related to the individual radiosensitivity status, which was observed in moderate radiosensitive humans. As shown in Figure 1d, SF2 values as it was previously reviewed by Devic et al.[16] reflect moderately radiosensitive individuals. However, the results of our study indicate that being moderately radiosensitive does not necessarily lead to the occurrence of AR, so a more precise classification is needed. As none of the donors in this study was hyper-radiosensitive or hyper-radioresistant, there is no evidence of how AR induction would be happened in these radiosensitivity levels. Nevertheless, Seong et al.[17] have reported the same AR incidence among three cell lines, independent from their different radiosensitivity statuses. In the above-mentioned study, because there was a limited number of cell lines that represent various radiosensitivity levels, the results seem to be insufficient for reaching a conclusion on the relationship between the AR phenomenon and the wide range of radiosensitivity. Although there is still no definite proved mechanism for AR occurrence, DNA repair possibility is assumed.[4,18] Also, individual radiosensitivity is probably affected by DNA repair mechanisms.[19] Therefore, the present study focused on the relationship between these two phenomena to investigate the induction pattern of RAR among human beings. Radiosensitivity was found to be affected by genetic capacity of an individual for repairing DNA double-strand breaks (DSBs) caused by IR.[20] Furthermore, inverse relationship between SF2 values and unrepaired DSBs was also determined.[21] Berthel et al.[15] have stated that the excess amount of ATM protein monomers because of LDIR can result in better recognition of DNA damages that are created after HDIR in moderate radiosensitive cases, as the function of AR. However, the radioresistant individuals by producing enough ATM monomers after being exposed to HDIR require no additional amount of this protein. Furthermore, it has been shown that RAR is influenced by DNA repair mechanisms. In this regard, Shelke and Das[4] have observed the AR occurrence through DNA damage in 16 individuals out of 20, and then reported the increased proteins’ expression of NHEJ DNA repair pathway in AR induction. Furthermore, it was demonstrated that transcription levels of some genes (KU70, KU80, DCLRE1C, and PRKDC) were higher in AR+ cases. Toprani and Das[5] have also reported the AR incidence among 12 donors out of 20 as well as the influence of BER pathway’s genes on AR phenomenon. It was shown that OGG1, XRCC1, and LIGASE3 genes were statistically up-regulated and only in AR+ group of individuals. However, sufficient data in all repair genes expression levels among AR+ and AR– populations based on their inter-individual differences still remain to be studied in future. Our findings have also indicated the influence of intrinsic radiosensitivity on the RAR. From the perspective of AR, normal individuals with relatively higher intrinsic radiosensitivity better react to the HDIR following a low priming dose. In the present study, the greatest decrease in chromosome aberrations frequencies was detected in the individual who had the highest radiosensitivity level, and conversely, the least chromosome aberrations reduction was observed in donor with the highest surviving fraction, who was the most radioresistant individual in the study. As demonstrated in Table 1, the first donor has the most chromosome aberrations after receiving a high dose of 2 Gy. However, the amount of these aberrations in the adapted samples (LD + HD) of this donor was 0.273, which is equal to those raised from the high-dose irradiation in seventh donor. Thus, it can be concluded that for a relatively radiosensitive individual, a priming low-dose irradiation can lower chromosome aberrations resulted from the subsequent high dose to the level of aberrations because of the same high dose of a relatively radioresistant individual. However, Komova et al.[22] have reported the independency of AR from intrinsic radiosensitivity by the endpoint of chromosome aberrations. Furthermore, it was assumed that the radioresistance following a hyperradiosensitivity to LDIR hyper-radiosensitivity/induced radioresistance (HRS/IRR), which occurs in a specific low-dose range for every single donor, is subjected to the same mechanism of AR. Thus, AR was expected to be induced after being exposed to the specific priming low dose for different individuals. It was also shown that there is no relationship between priming low-dose radiosensitivity in humans and the AR phenomenon. However, it is notable that HRS/IRR was not a constant response in all individuals.[23] Therefore, the results of this study could not be generalized to the population. It should be noted regarding the use of HRS in the pulsed low dose-rate radiotherapy, inter-individually variations may effect on clinical benefits of this method. Regarding the increased radioresistance of blood lymphocytes, because of the low-dose exposure of radiation before the subsequent higher dose, it is suggested that this resistance is better shown in those individuals who are inherently more radiosensitive compared to those with higher radioresistance and more intrinsically DNA repair capability.[24,25] Today, growing use of image-guided radiotherapy (IGRT) that use the computerized tomography (to position and monitor tumor response) with LDIR less than 100 mGy has increased the need for more focus on RAR during treatment. Utilizing such imaging procedures appears to be capable of modifying treatment results by increasing tumor cell survival through RAR.[26] Therefore, the evidence from this study suggests that this deleterious effect should be given more attention among radiosensitive individuals. CONCLUSION RAR, as a radio-protective phenomenon, does not occur consistently in all human beings. Its induction depends on several factors such as intrinsic radiosensitivity. Therefore, it can be concluded that, among normal individuals with moderate radiosensitivity status, AR is better recognizable in those individuals with relatively higher radiosensitivity. This radiosensitivity-dependent AR has the potential of affecting outcomes of some radiotherapy techniques such as IGRT. Because the number of donors and their intrinsic radiosensitivity statuses were limited in this study, further studies are needed to confirm our results. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship This work was supported by the Mashhad University of Medical Sciences. Conflicts of interest There are no conflicts of interest.

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