Effects of low-dose gamma radiation on DNA measured using a quartz tuning fork sensor

2.1 The human DNA model

DNA, located in a cell’s nucleus, is composed of nucleotides, each consisting of a phosphate group, a sugar molecule, and one of four nitrogenous bases: adenine (A), thymine (T), guanine (G), and cytosine (C). These bases form the genetic code, providing instructions for the cell’s development, functioning, and reproduction (de la Fuente Rosales, 2018). Genes, which are sequences of these bases, are the cell’s language, directing protein production. Nucleotides bond together to create two intertwined strands, forming a double helix structure, where adenine pairs with thymine and guanine pairs with cytosine. DNA coils into chromosomes, which contain DNA sequences in a cell. Humans have twenty-three pairs of chromosomes in each cell’s nucleus (Qasim and Ahmed, 2020).

2.2 Biological effects of radiation

Free radicals, such as hydroxyl radicals and superoxide anions, are highly reactive compounds characterized by an unpaired electron. Ionizing radiation, particularly photon irradiation, triggers water radiolysis, producing free radicals that can indirectly damage DNA. Reactive compounds, including hydrogen peroxide and hydroxyl ions, are also generated (Moreels, 2020). Radiation affects cells through two proposed mechanisms: direct and indirect, as shown in Fig. 1 (Gong et al., 2021). Depending on the cumulative energy transferred to a material, this energy may be high (as in alpha rays) or low [as in long-distance energy transfer (LET) (beta and gamma rays)] (Chatterjee and Walker, 2017). Through the direct effect, gamma rays can transmit both low and high energy via LET. They collide with atoms or molecules, dislodging electrons and inducing ionization. This can cause DNA fractures, including breaks in phosphodiester bonds or the opening of purine rings, affecting single- or double-stranded DNA. However, because ∼ 80 % human body cells comprise water, the probability of direct DNA interaction is low when exposed to ionizing radiation because DNA constitutes only 1 % of the cell (Wang, 2019). Approximately 65 % radiation-induced DNA damage is attributed to indirect effects caused by hydroxyl radicals. In this process, radiation ionizes molecules, producing free radicals that interact with DNA, leading to toxic effects and changing its chemical properties (Budak, 2020).

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Fig. 1

Mechanisms of IR: direct interaction damages DNA, while indirect effects generate free radicals from cellular molecules such as water, resulting in DNA damage (Gong et al., 2021).

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Equation (1) illustrates the disintegration of water under radiation (radiolysis), which creates four free radical products: H•, OH•, H⁺, and OH⁻:

Based on this reaction, free radicals from rapid structural breakdown interact with nearby molecules, leading to DNA damage such as cross-linking and DSB (McKee, May 2019). These destructive reactions activate DNA repair mechanisms, which typically effectively address single-strand breaks. However, repairing DSB is more challenging and can lead to errors during rejoining, such as mutations, chromosomal aberrations, or cell death (Kaleli-Can, 2021; Budak, 2020). DSBs are crucial for assessing radiosensitivity and quantifying chromosomal abnormalities following radiation exposure, making them valuable biological dosimeters (Moreels, 2020).

2.3 QTF sensors

QTFs represent a significant advancement in sensor technology for measuring mechanical, electrical, optical, and thermal parameters (Zhang, 2018). They find broad applications in chemistry and biology (Voglhuber-Brunnmaier, 2019), including acoustic microscopy (SAM), dynamic force spectroscopy, and scanning electron microscopy for high-resolution topographic analysis (Alshammari, 2020). QTFs are also integral to photothermal–acoustic spectroscopy for bioassays, petrochemical analysis, and environmental studies. Additionally, quartz-enhanced photoacoustic spectroscopy is used for gas sensing. Despite the inherent stability of QTFs owing to their physical characteristics, environmental conditions such as temperature, pressure, humidity, and gas density can experience rapid fluctuations (Rousseau, 2019).

Specifically, numerous experimental results confirm the exceptional sensitivity of QTFs to radiation, rendering them ideal for monitoring low-dose radiation in small rooms and laboratories. High levels of gamma irradiation can induce shifts in the resonance frequency of quartz resonators (Alanazi, 2022). For example, Shimoda et al. (Shimoda and Uno, Jan. 1988) explored the relationship between frequency shifts and gamma radiation, while Alanazi et al. (Alanazi et al., 2021) exposed gold-coated QTFs to gamma radiation from a cesium-137 source. They assessed the sensitivity of QTFs to radiation at various time intervals (0, 1, and 2.5 h) under consistent ambient conditions, revealing increased frequency shifts, with resonance frequencies increasing from 31 to 34.5 kHz for the QTFs. Alanazi et al. (Alanazi, 2023) also showed the reliability of QTFs in evaluating the enhancement of DNA damage at different concentrations of gold nanoparticles (GNPs) during irradiation, highlighting potential biomedical applications. Introducing GNPs during irradiation resulted in higher fork displacement frequencies, suggesting substantial enhancement in damage ratios. At 15 μg/mL, radiotherapy efficacy was significantly enhanced, underscoring the potential for integrating GNP-mediated radiosensitization with sensing technology.

This study employed the Quester Q10 commercial instrument (Fourien Inc., Canada), known for its high quality factor piezoelectric transducer consisting of a QTF with two vibrating prongs made of quartz. The 300 nm thick prongs are coated with silver electrode films and measure 3.73 mm in height and 0.52 mm in width, with a 0.3 mm gap between them (Fig. 2 (Alanazi et al., 2021). Equipped with an impedance analyzer, analog-to-digital converters, amplifiers, a controller, and a signal generator capable of conducting frequency sweeps, we recorded the QTF’s impedance response at a sampling rate of 0.5 million samples per second. The data acquisition system provided results in a comma-separated value format, which we analyzed using MATLAB, Origin Lab, and Python. Further system details are described in a previous study (Alodhayb, 2020). With the assistance of a piezoelectric material, quartz forks responding to a mechanical signal transform it into electrical excitations, resonating at frequencies of 32,758 Hz under vacuum conditions. When stimulated by a self-excitation setup, the forks exhibit lateral movement. The applied driving voltages to the QTFs during excitation range from 0.1–0.2 V. Consequently, introducing external mass loading is expected to affect the characteristic resonance frequency of QTFs, resulting in a damping effect on the oscillation frequency. However, increasing the mass on the quartz prongs of QTFs should cause a more significant negative shift in resonance frequency (Kaleli-Can, 2021).

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Fig. 2

Schematic illustrating a standard QTF sensor (Alanazi et al., 2021).

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2.4 Damage and repair mechanisms

DSBs in DNA are repaired by two primary processes: homologous recombination (HR) and nonhomologous end-joining (NHEJ). HR is error-free and occurs during the S and G2 phases, using the sister chromatid as a template. Error-prone NHEJ operates at any cell cycle phase (Duncan and Swensen, 2016). Specific molecules identify and repair altered DNA structures induced by IR triggered by specific genes. A damage checkpoint recognizes changes and activates pathways to protect the genome, known as DNA damage repair (Jia, 2021). Minor lesions, single-strand breaks, or noncomplex DSBs are repaired through nucleotide excision repair, base excision repair (BER), and NHEJ (Yousefzadeh et al., 2021).

Ataxia telangiectasia mutated (ATM), located on human chromosome 11q, plays a crucial role in various cell cycle checkpoints (G1–S, S, and G2–M). It is essential for identifying and repairing DNA damage. Minor DNA damage from low-dose radiation can typically be repaired with ATM, restoring function. However, high doses can cause severe denaturation, leading to challenging-to-repair DNA DSBs and potential cancer risks (Jia, 2021). A study assessing phospho-S1981 ATM levels in control cells demonstrated a significant increase just 15 min after 2 Gy irradiation, initiating repair signaling shortly after DNA damage induction (Blimkie, 2014). Yang et al. observed the dynamic repair process in human hepatocytes after 48 h of gamma-ray irradiation. They observed an increase in both DSBs and SSBs proportionate to the irradiation doses, with DSBs being more prevalent. After 24 h, partial repair occurred, covering up to 15 % chromatid DNA breaks and nearly 50 % stained chromatids. Repairing isochromatid breaks remained challenging (Jia, 2021). If damage signals persist despite initial repair mechanisms, cells do not replicate damaged DNA and prioritize mechanisms such as cell death, cell cycle arrest, or senescence (Yousefzadeh et al., 2021). The transcriptional regulator p53, activated by ATM, coordinates these responses by interacting with signal transduction and cell cycle regulation pathways. In severe DNA damage that is difficult to repair, cells can activate checkpoints and initiate apoptosis (van de Kamp, 2021).

(1) HR repair.

This modeling method facilitates the accurate repair of DSBs through NHEJ. At the same time, HR involves obtaining the correct sequence from intact DNA or initiating the resection of damaged DNA ends by nucleases to restore strand integrity. CtIP and Mre11 proteins are crucial for generating single-stranded DNA from DSB ends (Santivasi and Xia, 2014). The primary, slower DSB repair pathway during the S and G2 phases uses an undamaged sister chromatid as a repair template, ensuring error-free DNA restoration. Additionally, the HR pathway identifies DNA damage, activating the Mre11-Rad50-NBS1 (MRN) complex and ATM through BRCA1 proteins. ATM phosphorylates and activates BRCA1, initiating cell cycle checkpoints. Subsequently, BRCA1 indirectly interacts with Rad51, facilitating the exchange of damaged strands and their pairing with a homologous template to locate sequences, ultimately restoring the original double strand by elongating and ligating the broken strands (Penninckx, 2021).

(2) NHEJ repair.

NHEJ is an alternative repair pathway for various DSBs, though it exhibits lower fidelity than HR. Using Ku70 and Ku80 proteins (XRCC5), NHEJ protects damaged DNA from exonuclease enzymes and recombines DSB ends without requiring homologous sequences. This method ensures accurate repair without a homologous template. Ku70 and Ku80 identify DSBs and recruit proteins to the breakpoints, forming an active DNA-PK holoenzyme. Nucleases are subsequently recruited to expose DNA ends and digest nucleotides from both strands. NHEJ is a DNA repair system and a checkpoint activator (Iin and Yanti, 2015). Eventually, a stable complex involving DNA ligase I is formed, which binds the two repaired DNA ends facilitated by enhanced DNA-PK activity. This enhancement increases resistance to IR in various cancer cells, including glioblastoma multiforme (GBM), the most common primary brain malignancy. Therefore, leveraging a tumor’s deficiency in NHEJ for treatment planning may be a viable strategy (Penninckx, 2021).

3.1 Before the irradiation stage

Initially, pure DNA samples were prepared using commercial genomic DNA obtained at 2-ng/μL (60 μL in total) concentration, purchased from Applied Biosystems Company. Additionally, DI water and quartz tuning forks were acquired from FOURIEN (Edmonton, AB, Canada), as depicted in Fig. 2. Subsequently, a second sample was prepared by mixing pure DNA with deionized water to create a DNA solution (60 µL in total). Finally, a sample containing only water (60 µL) was prepared. Subsequently, the experimental setup was modified by removing the hermetic casing of the QTF to improve contact with the sample and observe DNA behavior. During sample recognition, the fork functioned normally, facilitating signal transduction from the conventional biosensors in the form of frequency shifts in each sample over 20 min. Fig. 5 shows a slight increase observed in the investigated samples.

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Fig. 5

Frequency shifts of DNA with and without water, as well as water alone, monitored over a 20-min period before exposure to gamma radiation, with measurements taken every 5 min.

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3.2 During the irradiation stage

In this study, the samples were exposed to radiation from a synthetic radioactive nuclide, cesium-137, which has a half-life of 30.2 years and a current activity of $5\mathrm{\mu }\mathrm{C}\mathrm{i}$ . The gamma source was positioned directly adjacent to the QTFs at a distance of 1 cm. The dose rate was ∼ 1.5 μGy/min, generating gamma rays with an energy of 662 keV. At different time intervals corresponding to various doses, the samples were exposed at room temperature (25 °C) in the chamber for 20 min. The samples were positioned 1 cm away from the cesium-137 source, parallel to the direction of the gamma source, as depicted in Fig. 3. During this phase, the fork was immersed in the samples, after which we exposed the samples to gamma radiation from a cesium-137 source. We observed deformation owing to DNA damage and investigated the effects of gamma radiation on DNA strand breaks in our samples. The baseline level of DNA oxidative damage was determined by calculating the difference in frequency moments before and after irradiation at 20 min, represented as the sensitive site of the fork frequency shift.

Fig. 6 shows the DNA damage frequency spectra results with and without water, including results for water alone. The results demonstrate an increase in frequency from ∼ 15 µGy to ∼ 22.5 µGy at an exposure time of ∼ 10 min; further exposure for 20 min increased the frequency to ∼ 30 µGy. Notably, the amount of DNA damage increased after ∼ 5 min of exposure: 7.5 µGy in the order of water, DNA, and DNA with water, resulting in $\mathrm{\Delta }f$ values of ∼ 16.71, 17.83, and 14.7 Hz, respectively. However, after 10-min irradiation with doses of 7.5–15 µGy, the $\mathrm{\Delta }f$ observed were ∼ 22.34, 24.05, and 15.21 Hz for water, DNA, and DNA with water, respectively. Finally, after 20 min (dose = 7.5–30 µGy), the frequency shifts were ∼ 39.21, 28.37, and 41.23 Hz for water, DNA, and DNA with water, respectively. Furthermore, a substantial increase in DNA strand breaks was observed, as indicated by the shifts in fork frequencies, with absorbed doses starting at 7.5 µGy in 5 min, increasing to 15 µGy during exposure, and peaking at 30 µGy.

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Fig. 6

DNA strand breaks induced by gamma irradiation without and with water over a 20-min exposure period. (a) From 20 to 40 min, with measurements recorded every 5 min, and (b) the progressive response of each sample with frequency shifts upon exposure to gamma radiation.

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Moreover, Fig. 6 and Table 1 show the recorded frequency shifts, highlighting a clear correlation between dose increments and observed frequency shifts during radiation exposure, reflecting the extent of DNA’s response to radiation. Thus, by monitoring the fork’s frequency behavior in DNA damage during exposure, we conducted experiments using several media: DNA alone, DNA combined with deionized water, and just water under identical conditions. According to Table 1, variations in $\mathrm{\Delta }f$ were observed, attributed to increased DNA damage.

After 10 min of exposure, the damage observed for water showed a frequency shift ranging from $\mathrm{\Delta }f$  ∼ 16.7 to 39.2 Hz. The frequency shift for DNA in aqueous solution ranged from $\mathrm{\Delta }f$  ∼ 14.7 to 34.4 Hz, indicating a noticeable increase. Therefore, this specific dose was selected as the maximum experimental dose. Consequently, in the presence of water, the DNA’s response to irradiation significantly increased, with the frequency increasing from $\mathrm{\Delta }f$  ∼ 28.37 to 41.23 Hz, nearly doubling its initial value. The examinations further indicated that, while the sample experienced significant frequency changes, these changes were comparatively less pronounced than those observed when the DNA was present alone. Consequently, in the presence of water, the DNA’s response to irradiation significantly increased, with the frequency increasing from $\mathrm{\Delta }f$  ∼ 28.37 to 41.23 Hz, approximately doubling its initial value. The examinations further indicated that, while the sample experienced significant frequency changes, these changes were less pronounced than those observed when the DNA was present alone. We attributed the shift in frequency to the gamma-ray interaction with water. In these interactions, water demonstrates indirect effects by generating highly reactive free radicals—H•, OH•, H⁺, and OH⁻. These radicals disperse widely, causing more extensive DNA damage. This increase in frequency was observed after 10 min of exposure, suggesting that indirect reactions are more damaging compared to direct exposure of pure DNA. Furthermore, no repair was observed in the presence of DNA alone or with water based on this observation. This finding underscores the need to discuss this in the following stage.

3.3 DNA repair stage

In the final stage, after the completion of the irradiation process as described earlier, the cesium-137 source was removed from the chamber after a 20-min exposure. This step was taken to observe the repair of DNA once the effects of radiation subsided. The fork was immersed in the same samples without the gamma radiation source, and the frequencies were recorded for DNA following exposure to gamma radiation doses ranging from 7.5 to 30 µGy. The findings indicated an initial increase in frequency expression levels immediately after exposure, followed by a decline in DNA with water at 60 min. Subsequently, DNA and water exhibited an increase in frequency shift, Δf ∼ 79 Hz and Δf ∼ 98 Hz, respectively, as detailed in Table 1. As depicted in Fig. 7, the residual damage was assessed immediately after irradiation (20 min) for each damage period and plotted against the postirradiation time. However, the frequency shift remained stable for water at ∼ 39.21 Hz for 10 min, increased to ∼ 41 Hz, and then decreased to ∼ 32 Hz. This shift is attributed to gamma rays impacting the water. The observed increase in frequencies of direct and indirect repair reactions indicates that gamma radiation caused complete DNA damage, which was challenging-to-repair owing to the lack of a suitable environment. This finding suggests that the DNA repair context did not significantly influence DNA responses in this study. However, the roles of DNA damage response proteins, such as gH2AX, ATM, 53BP1, RAD51, and the MRE11/RAD50/NBS1 complex at DNA damage sites, could greatly influence interpretations, particularly in genes related to recombination near DSB. This influence contributes to the repair mechanisms under investigation. Despite processing via recombination through BER enzymes in living cells, certain damages may persist, potentially leading to significant genetic consequences such as the induction of mutations. Indeed, there is mounting evidence that deficiencies in DNA repair stem from insufficient DNA damage response proteins. We performed a comparable experiment using water as a medium to investigate DNA damage response and repair under the ex vivo conditions used in this study. Water was irradiated under identical conditions as DNA, followed by removal from the radiation source to observe fork shift frequencies. Initially, the frequency increased upon exposure and subsequently decreased during the repair phase. These observations are indicators of damage and repair. Thus, our findings provide a biological rationale for the substantial increase in DNA damage predicted by a dose–response relationship and assessed through a sensitive assay.

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Fig. 7

Frequency shifts of DNA with and without water, and in just water, following exposure to gamma radiation. (a) Between 40 and 60 min, with measurements recorded every 5 min, and (b) the gradual response of each sample with frequency shifts after exposure to gamma radiation.

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