Introduction
One way to prime the body before a larger dose of radiation is to use low-dose ionizing radiation (LDIR), which is defined as those that do not exceed 100 mGy of low-LET radiation according to the classification provided by UNSCEAR1 In the study by Ito et al.2, a mid-lethal challenging dose of X-ray (5.9Gy) applied to 8-week-old C57BL/6 mice twenty-four hours after a 50 mGy priming dose. They reported an increase in the survival rate of mice compared to irradiated control. The pKZ1 transgenic mouse model has been previously used to measure dose response for DNA inversions in spleen and prostate for a wide dose range. Curiously, dose as low as 0.001 mGy completely protected against chromosomal inversions induced by a single high dose of 1Gy, and, also, against a proportion of spontaneously induced inversions3. In cancer-prone animals with a partial TP53 function, a single whole-body dose of 10 mGy does increases cancer latency and effectively restores a portion of the life that would have been lost due to either spontaneous or radiation-induced cancer in the absence of the low dose4. Increasing the priming dose to 100 mGy has imaginarily eliminated the protective effects. In other words, 100 mGy exceeded the upper dose threshold for induction of adaptive protection. Conversely, thymocyte apoptosis induced by a challenge dose of 2Gy was significantly decreased when the mice were preirradiated with a 100 mGy priming dose 6h prior to the challenge dose5, hence upper threshold dose appear to be higher than previously considered. An epidemiological study shows that no excess cancers have been found at doses below 200 mGy6, leading to the definition of low dose as 200 mGy or less7. However, from a radiation therapy view, doses in the range of 0.5–0.7Gy are defined as a low8. Exposure to an X-ray dose of 500 mGy did not render human lymphocytes refractory to chromatid deletions caused by a challenge dose of 1Gy9. Similar observations were reported by Broome et al.10 using normal human fibroblasts. By contrast, Wistar rats have recently been found to express lower MDA and NO levels after total-body irradiation with a dose of 5Gy if pre-treated several hours earlier with low dose of the order of 0.5Gy11. Conflicting findings from both sides of the radiation hormesis argument indicate that the exact effects at so-called “low dose” in radiation therapy view are not known. In the current study we investigated whether the pre-exposure to 0.5Gy as a priming dose can protect from the damaging effects of subsequent high one (2Gy). A variety of endpoints at different times following the high dose exposure were measured: mitotic catastrophe after 3 days; DNA damage, cell cycle redistribution, and apoptosis at earlier time points, 1 to 24h post-exposure. Whether adaptive response could be induced in tumor cells by LDIR has likewise remained elusive. Studies have revealed that tumor cells are resistant to LDIR-induced adaptive response12 or showed a distinct pattern from normal cells13. Cultures pre-exposed to 75 mGy followed by 4Gy at a 12-hour interval had fewer surviving cells than those exposed to 4Gy alone, indicating that the predose significantly enhanced the tumor-killing effect of the subsequent high one13. This was confirmed in vivo using S180-bearing mice14. Therefore, the present study was also aimed at determining the ability of 0.5Gy to induce an adaptive response in A549 cells to a subsequent challenging dose of 2Gy.
Materials and methods
Cell line and cell culture
Human cancer-derived cell A549 and normal fibroblast called human embryonic fibroblast, lung-derived cell line (WI-38) were obtained from VACSERA -tissue culture unit (Giza, Egypt). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 1% antibiotic-antimycotic solution (10,000 units/ml of penicillin, 10,000µg/ml of streptomycin, and 25µg/ml of amphotericin B) and 10% fetal bovine serum (FBS, Gibco). Cells were grown in 25–75cm culture flasks and maintained in a humidified 37°C incubator with 5% CO2.
Irradiation
Cells were exposed to γ-radiation doses of 0–0.5Gy 24h before being exposed to a challenge dose of 2Gy. The irradiation procedure was performed at the Atomic Energy Authority, Cairo, Egypt, using the Canadian gamma cell-40 (137Cs) at a dose rate of 0.333Gy/min. Cells were irradiated under aerobic conditions at room temperature and returned to the incubator between priming and challenge dose exposures.
Comet assay
Cells were harvested 1 h post exposure to 2 Gy γ-irradiation and then subjected to the comet assay in alkaline solution using the Comet Assay Kit (Abcam, ab238544) according to the manufacturer’s protocol. Briefly, ∼ 105 cells were combined with Comet Agarose at a 1:10 ratio and immediately transferred onto the slide glasses covered with a comet agarose base layer. Slides were stored at 4°C in the dark for 15min before being immersed in lysis buffer for 60min. This was then followed by immersion in an alkaline unwinding solution for 30min. Slides were inserted into the electrophoresis chamber, immersed in the buffer, and an electric potential of 1V/cm was applied for 20min. After electrophoresis, the slides were rinsed three times (each for 5 min) with deionized water, followed by a single wash with 70% ice-cold ethanol, and then dried at room temperature. Once dry, 100µl/well of 1× Vista Green DNA Dye was added, and microscopy images were taken using the Labomed LX400 fluorescent microscope. Comet tail moments were analyzed using the CometScore™ software from TriTek Corporation. Cells analyzed were categorized into three DNA damage groups based on their tail moment values. Cells with a tail moment less than 2 were classified as undamaged (comet type 1); cells with a tail moment of 2 or higher were considered damaged (comet types 2, 3, 4, and 5). Among damaged cells, those with tail moment values exceeding 30 were designated as apoptotic (comet type 5b).
Apoptosis analysis
Apoptosis was quantitated using the FITC Annexin V/Dead Cell Apoptosis Kit (Invitrogen, Cat# V13242) according to the manufacturer’s instructions. Briefly, after 24h from 2Gy exposure, cells were harvested by trypsinization and washed with cold PBS. After centrifugation (1000rpm, 5min), cell pellets were resuspended in 100 µL 1× annexin binding buffer. Subsequently, 5 µL of FITC Annexin V and 1 µL of a propidium iodide solution (100µg/mL) were added. After a 15min incubation at room temperature, the mixture was diluted by adding 400 µL of 1× annexin binding buffer. Acquisition and analysis were performed on a Navios Flow Cytometer (Beckman coulter life science, USA) using the Navios software.
Cell cycle analysis
Cell cycle status was determined using Vybrant™ DyeCycle™ Violet Stain (Thermo Fisher Scientific, Cat# V35003) according to the manufacturer’s protocols. Cells were collected 24h after 2Gy irradiation by trypsinization, fixed in ice-cold methanol and stained with the DNA-selective stain Vybrant DyeCycle Violet. Eventually, the stained cells were analyzed on a Beckman Coulter Navios flow cytometer with excitation at 405nm and emission measured at 440nm.
Mitotic catastrophe
Cells were seeded on sterile coverslips placed in four-well plates. After overnight attachment, cells were irradiated as indicated. Subsequently, Cells were fixed 72h after the final radiation dose (2Gy) using a 4% paraformaldehyde solution in 1× PBS for 20min at room temperature. After one PBS wash, coverslips were mounted, cell side down, onto labelled microscope slides using ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher, Cat# P36935). Nuclear morphology was examined under a fluorescence microscope (Labomed LX400). Cells containing nuclei with two or more distinct lobes or aberrant nucleus morphology were scored as undergoing mitotic catastrophe15.
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cells 24h after exposure to 0.5–2Gy using the Qiagen RNeasy Mini Kit (Cat# 74104) according to the manufacturer’s protocol, and subsequently reverse transcribed to cDNA with the QuantiTect Reverse Transcription kit (Qiagen, Cat# 205310). Thermal cycling parameters for the reverse transcription reactions were: 15min at 42°C, then 3min at 95°C, followed by holding on ice. qRT-PCR was performed on a 5 plex Rotor-Gene PCR Analyzer (Qiagen) using SYBR Green PCR Master Mix (QuantiTect SYBR green PCR kit, Cat# 204141) and the following QuantiTect primers: HMOX1 (QT00092645), BCL2 (QT00025011), CDK6 (QT00019985), and WEE1 (QT00038199). Gene Expression was normalized to the housekeeping gene, ACTB (QT000954231), and relative gene expression was determined using the 2−ΔΔCt method.
Statistical analysis
All experiments were independently performed at least twice, and intergroup comparisons were analyzed using unpaired two-tailed Student’s t-tests. A P value below 0.05 was deemed statistically significant.
Results
The 0.5Gy priming dose conferred WI-38 human lung fibroblasts a resistance to DNA damage induced after a large 2Gy challenge dose
DNA is the main cellular target damaged by exposure to ionizing radiation. Comet assay under alkaline condition helps in quantitative assessment of DNA damage. Tail moment, which is the product of tail DNA and tail length, was the parameter used to express the comet assay results. In WI-38 cell, treatment with a challenging dose of 2Gy led to an increase in tail moments as compared to non-treated controls (Fig.1). However, the magnitude of the increase in cells treated with both predose and challenging dose was less than that in those treated only with the challenging dose. These findings indicate that the cellular response to the challenging dose of radiation differs when cells are primed with 0.5Gy.
A priming dose of 0.5Gy protected lung fibroblasts from DNA damage caused by a subsequent 2.0Gy dose administered 24h later. Representative images (a) and quantification (b) of tail moments in WI-38 cells at 1h post 2Gy irradiation. Thirty cells at least were analyzed in each group. Quantification is represented as values expressed as fold change relative to control (Ctrl). (c) Classification of DNA damage. A bar graph was used to show the distribution of cells among three DNA damage levels. Two independent experiments were conducted. The level of significance was represented as *P < 0.05, Student’s t-test.
Comets analyzed were classified by tail moment values to differentiate normal, damaged, and apoptotic cells, as described by Genghini et al.16. In cultures treated solely with the challenging dose, 45% of comets had tail moment values indicating damage, with 20% being apoptotic. In contrast, cultures treated with predose + challenging dose showed 33% of values indicating damage with 5% being apoptotic (Fig.1c).
The 0.5Gy priming dose partially reversed the challenge dose-induced G0/G1 arrest and apoptosi
Following DNA damage, Cells either pause cell cycle progression until errors have been corrected or undergo apoptosis if the damage is massive and irreparable. In mock-irradiated controls, the cell-cycle phase distribution was 12% in G0/G1, 4% in S, and 84% in G2/M phase (top row, left panel of Fig.2a). After a high dose exposure (2Gy), WI-38 cells experienced a delay in moving out of G1 and into S phase, resulting in a cell cycle distribution of 99% in G0/G1, 0.8% in S, and 0.2% in G2/M (top row, middle panel of Fig.2a). However, 0.5Gy pretreatment of lung fibroblasts attenuated 2Gy-induced G1 phase cell cycle arrest; released some cells from G1 arrest into S-phase (G0/G1:S: G2/M phases = 93%: 5%: 2%). Eukaryotic cells have about 20 cyclin-dependent kinases (CDK), but only CDK1-6 are related to the cell cycle. CDK4/6 specifically regulates cellular transition from the G1 phase to the S phase, where DNA synthesis occurs. Cells treated with the challenge dose alone showed lower CDK6 levels and higher Wee1 levels than those treated with the predose + challenge dose (Fig.2b), indicating increased DNA damage induction. It is worth mentioning that Wee1 negatively regulates entry into mitosis (i.e., the G2/M transition).
(a) Histograms obtained from flow cytometric analysis showing WI-38 cell cycle at 24h after challenge with a dose of 2Gy. (b) Reverse transcription-quantitative PCR results for the detection of CDK6 and Wee1 mRNA expression. (c) Representative flow cytometry dot plots of WI-38 cells after challenge with a dose of 2Gy. At 24h post-2Gy IR, cells were harvested, stained with Annexin V/PI and analyzed using a flow cytometer. Apoptosis is shown as values expressed as fold change relative to control (Ctrl). (d) Bar graph depicting the percentage of membrane-intact and membrane-damaged cells following 2Gy exposure, with and without a 0.5Gy priming dose. Data are means ± SEM (n = 2); *P < 0.05, **P < 0.01 and ***P < 0.001 by Student’s t-test.
The apoptotic rate in cells receiving the combination treatment was significantly lower than in those treated with the challenge dose alone (Fig.2c). Flow cytometry analysis showed that 82% of cells treated with the predose + challenge dose retained membrane integrity, compared to 70% in the samples treated solely with the challenge dose (Fig.2d).
0.5Gy pretreatment markedly attenuated late-type cell death induced by subsequent 2Gy exposure
Mitotic catastrophe is a delayed cell death that typically occurs 2–6 days after irradiation. Cells undergoing mitotic catastrophe die during mitosis. Mechanistically, erroneous repair of radiation-induced DNA breaks leads to chromosome aberrations that subsequently prevent accurate segregation during mitosis, cause defects in mitotic exit, and ultimately result in cell death. Mitotic catastrophe is characterized morphologically by distinct nuclear changes, including a multilobed or aberrant appearance (Fig.3a,b). DAPI staining revealed that 12% of cells treated solely with the challenging dose showed mitotic catastrophe, compared to only 7% in those treated with the predose + challenging dose (Fig.3c,d).
Representative images showing the nuclear morphology of (a) normal cells and (b) cells undergoing mitotic catastrophe. (c) Quantified data for mitotic catastrophe induced in cells treated with IR. 72h after the final radiation dose (2Gy), cells were fixed and stained with DAPI. Images of nuclei were acquired using a 40X lens. 100 nuclei were scored. Data are summary of two independent experiments and values are expressed in mean ± SEM. P values determined by two-sided Student’s t-test; **P < 0.01.
Molecular mechanisms underlying the hormetic effect of 0.5Gy ionizing radiation in normal fibroblasts
In WI-38, low-dose exposure of 0.5Gy significantly increased HMOX1 mRNA expression (Fig.4a). Heme oxygenase 1 (HMOX1, commonly HO-1) plays a cytoprotective role. In response to stress, HO-1 moves to the nucleus and, by interacting with other proteins, regulates the transcription of antioxidant and cytoprotective genes. This is strongly supported by the observation that the cytotoxic effects of hydrogen peroxide and other pro-oxidant agents are exacerbated in cells that lack HO-1 (Hmox1−/−)17. High doses of ionizing radiation, on the other hand, reduce cell antioxidant concentrations18. Mechanistically, the balance between reactive oxygen species (ROS) production and antioxidant defense shifts in favor of ROS. Here, a dose of 2Gy of gamma radiation clearly reduced levels of Superoxide dismutase (SOD), an important intracellular antioxidant (Fig.4b). However, SOD levels in the 0.5Gy + 2Gy irradiated cells were significantly higher than in the 2Gy irradiated cells (P < 0.01).
Changes in the (a) HMOX1, (b) SOD, (c) Bcl2, IL-1β, and TNF-α transcription levels in response to low dose irradiation. Twenty-four hours following exposure to radiation (0.5–2Gy), WI-38 cells were harvested, and target genes expression relative to β-actin were determined by real-time PCR. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s t-test.
Apoptosis occurs through two major pathways: extrinsic and intrinsic pathways. Intrinsic apoptosis, activated by DNA damage, is primarily regulated by B-cell lymphoma 2 (Bcl-2) family proteins, which compose of both anti- and pro-apoptotic members. The anti-apoptotic subfamily includes Bcl-2, Bcl-XL, Bcl-W, Bcl-2-A1, and MCL119. Exposure to 0.5Gy increases Bcl2 levels in WI-38 (P < 0.001, Fig.4c). On the other hand, the immune system regulates the extrinsic pathway of apoptosis. ROS generated from the higher challenge dose modulates cellular changes through non-nuclear interaction, increasing the transcription and subsequent release of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin-1β (IL-1β). These cytokines induce cell death via the death receptor pathway20,21. Significantly lower concentrations of IL-1β were detected after predose treatment. Oppositely, TNFα levels were not significantly altered (P > 0.05). However, WI-38 cells showed a tendency to release a less TNFα after irradiation with 0.5Gy (Fig.4c). The reduced production of these pro-inflammatory cytokines and upregulation of Bcl2 may have given WI-38 cells resistance to apoptosis induced by a 2Gy challenge dose.
Pretreatment with 0.5Gy protects lung cancer cells (A549) from apoptosis induced by subsequent 4Gy exposure
Indeed, previously published work on the response of A549 cells to LDIR is inconclusive. A 100-mGy priming dose has not been found to induce an adaptive response to a subsequent 6h later challenge dose22. Increasing the interval between doses to 24h did not produce different results23. However, raising the priming dose to 200 mGy was associated with protection against apoptotic death induced by a challenge dose of 20Gy24. In our study, Cells pre-exposed to a 0.5Gy “primed” dose before a 2Gy exposure showed a lower percentage of apoptosis than those exposed to the 2Gy alone (P < 0.05), indicating the presence of an adaptive response (Fig.5a). Figure5b show that ∼45% of cells treated with 0.5–2 Gy retained membrane integrity, whereas only 27% of those treated with the challenge dose alone did so. The molecular mechanisms of the hormesis phenotype involve antioxidant production and apoptosis pathways regulation, analogous to observations in normal cells. A 0.5 Gy pretreatment of lung adenocarcinoma cells significantly increased the mRNA expression of HMOX1, SOD, and Bcl2, which encodes intrinsic apoptosis pathway control protein. A549 cells also showed a marked reduction in the release of IL-1β (P < 0.05) and TNF-α (P < 0.01) following 0.5Gy ionizing radiation exposure (Fig.5c).
In A549, cell death induced after a large 2Gy challenge dose could be partly reversed by pre-treatment of cells with a dose of 0.5Gy. (a) Representative flow cytometry dot plots of A549 cells after challenge with a dose of 2Gy. At 24h post-2Gy IR, cells were harvested, stained with Annexin V/PI and analyzed using a flow cytometer. Apoptosis is shown as values expressed as fold change relative to control (Ctrl). (b) Bar graph depicting the percentage of membrane-intact and membrane-damaged cells following 2Gy exposure, with and without a 0.5Gy priming dose. (c) Reverse transcription-quantitative PCR results for the detection of HMOX1, SOD, Bcl2, IL-1β and TNF-α mRNA expression. Twenty-four hours after exposure to 0.5Gy, A549 cells were harvested, and target genes expression relative to β-actin were determined by real-time PCR. Data are means ± SEM (n = 2); *P < 0.05, ***P < 0.001, ****P < 0.0001 by Student’s t test.
Discussion
In this study we investigated the effects of exposing Lung adenocarcinoma A549 cells and normal lung fibroblasts WI-38 to ionizing radiation with or without administrating a 0.5Gy priming dose. Evidence indicates that the adaptive response is not immediately expressed, it requires a time-lag. Consequently, Cells were primed 24h before administration of the challenging dose. The 24-hour interval was chosen in accordance with previously published studies25. In WI-38, there was a statistically significant difference between mean normalized tail moments when comparing treatment with the challenge dose alone to treatment with 0.5Gy prior to the challenge dose (P < 0.05). Moreover, pretreatment with 0.5Gy reduced G1 phase cell cycle arrest and cell death—either through apoptosis or mitotic catastrophe—induced by the subsequent 2Gy exposure. These results confirm previous studies that indicate the so-called “low dose”, from the radiation therapy view, can induce resistance in mammalian cells to subsequent high doses. Of these, Otsuka report, where he and colleagues used the comet assay to analyze DNA damage in the spleens of C57BL/6N mice. They found that DNA damage induced by a 1.6Gy challenge dose was significantly reduced in the mice that had been preirradiated at 1.2 mGy/h for 23 days (0.5Gy in total) compared with that in the sham-irradiated mice26. A study on 30-day mortality found that mice pre-exposed to 0.5Gy at a rate of 0.3Gy/min had better survival rates than un-adapted mice after a subsequent exposure to 6.50Gy of carbon ions (290MeV/nucleon)27. Saini et al.28 investigated the expression levels of selected DNA damage response genes in G0 peripheral blood mononuclear cells (PBMCs) following irradiation. Blood samples from 25 healthy volunteers were exposed to 0.6Gy priming dose followed by a challenging dose of 2Gy, 4h later. The transcription levels of ATM, ATR, MDM2, GADD45A, and CDKN1A genes were not altered, while Cyclin E and CDK2 expressions were higher in cells pre-exposed to a 0.6Gy “adaptive” dose followed by the 2Gy exposure than in those exposed to the 2Gy dose alone.
Pre-exposing A549 cells to 0.5Gy significantly reduced apoptosis induced by the subsequent 2Gy dose, raising questions about previous findings on the differential responses of tumor and normal tissues to low-dose radiation23. Specifically, low-dose irradiation has been shown to protect normal lung epithelial cells from damage of a subsequent 5Gy challenging dose, likely through the activation of the ATM/AKT/GSK-3β pathway. However, this protective effect was not seen in A549 cells under similar conditions.
The mechanism by which 0.5Gy reduces vulnerability to subsequent ionizing radiation exposure is unclear. The study by Dieriks et al.25 aimed to investigate the adaptive response induced in human primary fibroblasts exposed to 0.5Gy of X-ray 24h before a 2Gy dose. The 0.5Gy priming altered the response of the DNA repair protein, phosphorylated histone H2AX (named γH2AX), and increased secreted cytokine levels relative to non-primed cells. Interestingly, these effects were not replicated by priming the cells with cytokines IL-6 nor TGF-β, indicating the adaptive response is regulated by another signaling pathways. Here, HMOX1 expression was elevated in primed cells, where HO-1, encoded by the HMOX1 gene, interacts with Nrf2 to increase the expression of various genes involved in the detoxification and elimination of oxidative stress, including SOD. This increase in antioxidant expression mitigated cellular damage from reactive oxygen species induced by high-dose radiation. Additionally, cells responded to a priming 0.5Gy by increasing the expression of Bcl2 and decreasing of IL-1β and TNF-α. These changes may explain primed cells’ resistance to cell death induced by the subsequent 2Gy dose.
Collectively, the 0.5Gy priming help cells resist subsequent challenge dose and evade induced damages by modulating both HO-1/Nrf2 (antioxidant production) and apoptosis signaling pathways. Both normal and cancer cells have survived, posing a barrier to the clinical application of LDIR in cancer therapy.
Data availability
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.
References
UNSCEAR, Sources, Effects Risks of Ionizing Radiation. Report To the General Assembly Scientific Annexes AB. UNSCEAR 2012 Report. United Nations Scientific Committee on the Effects of Atomic Radiation. United Nations Sales Publication E.16.IX.1 (United Nations, 2015).
Ito, M. et al. Effect of low-dose total-body irradiation on transplantability of tumor cells in syngeneic mice. J. Radiat. Res. 49 (2), 197–201 (2008).
Day, T. K. et al. Extremely low priming doses of X radiation induce an adaptive response for chromosomal inversions in pKZ1 mouse prostate. Radiat. Res. 166 (5), 757–766 (2006).
Mitchel, R. E. et al. Low doses of radiation increase the latency of spontaneous lymphomas and spinal osteosarcomas in cancer-prone, radiation-sensitive Trp53 heterozygous mice. Radiat. Res. 159 (3), 320–327 (2003).
Gong, S. L. et al. Adaptive response of thymocyte apoptosis and cell cycle progression induced by low dose X-ray irradiation in mice. Biomed. Environ. Sci. 13 (3), 180–188 (2000).
CAS PubMed MATH Google Scholar
Heidenreich, W. F., Paretzke, H. G. & Jacob, P. No evidence for increased tumor rates below 200 mSv in the atomic bomb survivors data. Radiat. Environ. Biophys. 36 (3), 205–207 (1997).
Fry, R. J. M. A Note On The Dose-Rate-Effectiveness Factor and Its (2013). https://three.jsc.nasa.gov/articles/DDREF.pdf
Niewald, M. et al. Randomized multicenter follow-up trial on the effect of radiotherapy on painful heel spur (plantar fasciitis) comparing two fractionation schedules with uniform total dose: first results after three months’ follow-up. Radiat. Oncol. 10 (1), 174 (2015).
Shadley, J. D. & Wiencke, J. K. Induction of the adaptive response by X-rays is dependent on radiation intensity. Int. J. Radiat. Biol. 56 (1), 107–118 (1989).
Broome, E. J., Brown, D. L. & Mitchel, R. E. Dose responses for adaption to low doses of (60)Co gamma rays and (3)H beta particles in normal human fibroblasts. Radiat. Res. 158 (2), 181–186 (2002).
Hussien, S. M. Radio-adaptive response induced by Low-dose ionizing radiation in innate immunity for radiotherapy. Health Phys. 124 (3), 166–174 (2023).
Park, S. H. et al. Different induction of adaptive response to ionizing radiation in normal and neoplastic cells. Cell. Biol. Toxicol. 15 (2), 111–119 (1999).
Jiang, H. et al. Low-dose radiation induces adaptive response in normal cells, but not in tumor cells: in vitro and in vivo studies. J. Radiat. Res. 49 (3), 219–230 (2008).
Yu, H. et al. Different responses of tumor and normal cells to low-dose radiation. Contemp. Oncol. (Pozn). 17 (4), 356–362 (2013).
CAS PubMed MATH Google Scholar
Kobayashi, D. et al. One-step protocol for evaluation of the mode of radiation-induced clonogenic cell death by fluorescence microscopy. J. Vis. Exp., 128 (2017).
Genghini, R. et al. Determination of genotoxicity of classical swine fever vaccine in vitro by cytogenetic and comet tests. Mutagenesis 21 (3), 213–217 (2006).
Poss, K. D. & Tonegawa, S. Reduced stress defense in heme oxygenase 1-deficient cells. Proc. Natl. Acad. Sci.. 94(20), 10925–10930 (1997).
Nuszkiewicz, J., Woźniak, A. & Szewczyk-Golec, K. Ionizing radiation as a source of oxidative Stress—The protective role of melatonin and vitamin D. Int. J. Mol. Sci. 21 (16), 5804 (2020).
Yang, D. et al. Pharmacological targeting of Bcl-2 induces caspase 3-Mediated cleavage of HDAC6 and regulates the autophagy process in colorectal Cancer. Int. J. Mol. Sci. 24 (7), 6662 (2023).
Wang, P. et al. Role of signal transduction pathways in IL-1β-induced apoptosis: pathological and therapeutic aspects. Immun. Inflamm. Dis. 11 (1), e762 (2023).
Shakibaei, M. et al. Redox regulation of apoptosis by members of the TNF superfamily. Antioxid. Redox Signal. 7 (3–4), 482–496 (2005).
Wei, X. et al. Enhancement of the tumor suppression effect of High-dose radiation by Low-dose Pre-radiation through Inhibition of DNA damage repair and increased pyroptosis. Dose-Response 22 (2), 15593258241245804 (2024).
Yang, G. et al. Distinct biological effects of low-dose radiation on normal and cancerous human lung cells are mediated by ATM signaling. Oncotarget 7(44). (2016).
Wang, X. C. et al. The adaptive responses in Non-Small cell lung Cancer A549 cell lines induced by Low-Dose ionizing radiation and the variations of MiRNA expression. Dose Response. 19 (4), 15593258211039931 (2021).
Dieriks, B. et al. Repeated exposure of human fibroblasts to ionizing radiation reveals an adaptive response that is not mediated by interleukin-6 or TGF-β. Mutat. Research/Fundamental Mol. Mech. Mutagen. 715 (1), 19–24 (2011).
Otsuka, K. et al. Activation of antioxidative enzymes induced by Low-Dose-Rate Whole-Body γ irradiation: adaptive response in terms of initial DNA damage. Radiat. Res. 166 (3), 474–478 (2006).
Wang, B. et al. Relieved residual damage in the hematopoietic system of mice rescued by radiation-induced adaptive response (Yonezawa Effect). J. Radiat. Res. 54 (1), 45–51 (2012).
Saini, D. et al. Transcription profile of DNA damage response genes at G0 lymphocytes exposed to gamma radiation. Mol. Cell. Biochem. 364 (1), 271–281 (2012).
Acknowledgements
The author would like to thank Prof. Nashwa El-Khazragy for her helpful advice on various technical issues encountered while preparing this paper.
Funding
Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).
Author information
Authors and Affiliations
Biophysics lab, Physics Department, Faculty of Science, Al-Azhar University, Nasr city, Cairo, 11884, Egypt
Yasser F. Ali,Ibrahim M. Hassan&Hussein M. Abdelhafez
Radiation Physics Department, National Center for Radiation Research and Technology, Egyptian Atomic Energy Authority, Cairo, Egypt
Omar S. Desouky
Authors
- Yasser F. Ali
View author publications
You can also search for this author inPubMedGoogle Scholar
- Ibrahim M. Hassan
View author publications
You can also search for this author inPubMedGoogle Scholar
- Hussein M. Abdelhafez
View author publications
You can also search for this author inPubMedGoogle Scholar
- Omar S. Desouky
View author publications
You can also search for this author inPubMedGoogle Scholar
Contributions
All authors have accepted responsibility for the manuscript’s content, consented to its submission, reviewed the results, and approved the final version. OD and YFA designed the experiments, which were carried out by YFA, IMH, and HMA. YFA prepared the manuscript with contributions from all co-authors.
Corresponding author
Correspondence to Yasser F. Ali.
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Ali, Y.F., Hassan, I.M., Abdelhafez, H.M. et al. 0.5Gy confers resistance to a subsequent high dose of γ-rays by modulating HO-1/Nrf2 and apoptosis pathways. Sci Rep 15, 9199 (2025). https://doi.org/10.1038/s41598-025-91667-9
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-025-91667-9
Keywords
- Adaptive response
- 0.5 Gy
- HMOX1
- Low dose
- Apoptosis pathways
