XR are a form of ionizing radiation widely used in medical imaging, industrial testing, and research applications. During diagnostic procedures, both patients and healthcare personnel may be exposed to low doses of XR (<100 mGy). The ionization produced by this type of radiation can induce physical, chemical, and biological damage in living organisms. Historically, nuclear events such as those in Hiroshima, Nagasaki, and Chernobyl have revealed the devastating effects of ionizing radiation on human health, including skin burns, ocular lens damage, thyroid dysfunction, and an increased risk of cancer, particularly leukemia (UNSCEAR, 2020). The biological effects of ionizing radiation have been extensively studied for decades, with most research focused on high-dose exposures. Although the detrimental consequences of high-dose radiation are well established, the biological impact of low-dose XR exposure on chromosomal integrity remains less clear and continues to be a matter of debate, as contradictory findings persist (BEIR VII, 2006; WHO, 2023). Several studies have shown that low-dose XR can induce DNA double-strand breaks, MN formation, sister chromatid exchanges, and other chromosomal abnormalities, suggesting that even minimal exposure may compromise genome stability (Huang et al., 2003; Rothkamm and Löbrich, 2003). However, other investigations report negligible or transient effects, proposing the existence of efficient DNA repair mechanisms or adaptive responses that mitigate chromosomal damage at low doses (Tubiana et al., 2006). These inconsistencies highlight the need for more comprehensive and standardized studies to clarify the true chromosomal consequences of low-dose XR exposure. A deeper understanding of these subtle yet potentially cumulative effects is essential for accurately assessing long-term health risks and refining radioprotection strategies aimed at safeguarding both, patients and occupationally exposed individuals (INSST, 2025).

Several studies have documented the harmful health effects of XR exposure, leading to the establishment of annual dose limits for individuals with occupational exposure, such as nuclear power plant workers (ICRP, 2007). Notably, epidemiological evidence indicates that children exposed to multiple computed tomography scans show an increased risk of developing leukemia and brain tumors (Henry and Arcangeli, 2021).

Despite these findings, a substantial knowledge gap persists regarding the biological consequences of low-dose XR exposure. In particular, there is limited consensus on the types of chromosomal alterations most reliably induced at low doses (e.g., losses, gains, dicentrics, translocations, deletions, MN, or locus-specific breaks) and on their frequency, which appears highly variable across studies (Rothkamm and Löbrich, 2003; Smith et al., 2003). Inconsistencies arise from differences in detection methods, dose-rate conditions and cell types examined, making it difficult to determine whether low-dose XR induce chromosomal alterations detectable through sensitive cytogenetic assays (Bryant et al., 2010). This gap is particularly relevant given that the general population is continuously exposed to ionizing radiation, both, from natural sources, such as cosmic rays, terrestrial radiation, and radionuclides contained in minerals, and from artificial sources, including XR-emitting equipment used in diagnostic and therapeutic procedures (NCRP, 2009).

The limited understanding of the genetic and chromosomal risks associated with low-intensity exposures hinders accurate risk assessment and the development of effective radioprotection strategies, as most low-dose studies report general increases in cytogenetic damage without detailing the affected chromosomes, the predominant types of alterations, their clonal or non-clonal nature, or their frequency. This is particularly relevant because even low-dose exposures may generate cumulative genetic damage and increase the risk of chronic diseases, including cancer. Therefore, a detailed characterization of the type, frequency, and spectrum of chromosomal alterations induced by low-dose XR exposure is essential to better elucidate its biological effects and to support public health protection strategies.

In this context, the present study aimed to characterize the cytogenetic effects of low-dose XR exposure in human peripheral blood lymphocytes in vitro, focusing on the type and frequency of numerical and structural chromosomal alterations, including both clonal and non-clonal events, and the overall spectrum of genetic damage, using non-exposed samples as controls. Our findings demonstrate that even low-dose XR exposure can induce numerical and structural chromosomal alterations, increase MN frequency, and enhance chromosomal fragility. These results provide important insights into the biological consequences of low-dose radiation and underscore the need to strengthen biosafety measures and radioprotection strategies for individuals exposed to XR, even at doses traditionally considered safe.

Ionizing radiation is an unavoidable component of the human environment, arising from both natural and anthropogenic sources, with medical X-rays representing the primary source of artificial exposure for the general population (Karmaker et al., 2021; Jackson and Bartek, 2009; UNSCEAR, 2020). The widespread and increasing use of diagnostic and interventional procedures has made medical XR the dominant man-made contributor to annual effective dose in many countries (Betlazar et al., 2016; ICRP, 2007). Although typically delivered at low doses, XR exposure can induce subtle yet persistent molecular and cellular effects, particularly in radiosensitive systems such as the hematopoietic compartment, leading to genome instability, impaired DNA repair, and oxidative and inflammatory responses (Cherednichenko et al., 2024). Despite their extensive clinical application, the subclinical and long-term genotoxic effects of low-dose XR exposure remain insufficiently characterized, underscoring the need for early detection of genetic alterations to refine radiobiological risk models and strengthen radioprotection strategies (Gnanasekaran, 2021).

In this context, a substantial knowledge gap persists regarding the type and frequency of chromosomal alterations induced by low-dose XR. Although previous studies have shown general increases in cytogenetic damage at low exposures, they rarely provide detailed descriptions of which chromosomes are most affected, which structural or numerical alterations predominate, or how frequently each alteration occurs. To address this limitation, our study employed GTG-banding to perform an in vitro cytogenetic assessment focused on the characterization of radiation-induced chromosomal damage, enabling the visualization of both clonal and non-clonal chromosomal abnormalities and providing precise information on their frequency.

Our results demonstrate that low-dose XR exposure induces measurable genetic and chromosomal damage in human peripheral blood lymphocytes in vitro, as reflected by increased MN formation and higher frequencies of structural and numerical chromosomal alterations associated with chromosomal instability. These findings are consistent with radiation-induced disruptions of DNA integrity and chromosomal segregation, indicative of persistent genotoxic stress. Similar cytogenetic effects have been reported in occupationally exposed populations, such as interventional radiologists chronically exposed to higher cumulative doses of ionizing radiation (Kochanova et al., 2023). Although differences in exposure patterns preclude direct dose–response comparisons, our data show that a substantially lower acute dose (94.3 mGy) is sufficient to induce significant chromosomal alterations and fragile sites compared with non-irradiated controls, reinforcing the capacity of ionizing radiation to induce cytogenetic damage.

Importantly, our results suggest that measurable chromosomal instability can occur at doses well below those reported in occupational settings, underscoring the sensitivity of cytogenetic endpoints for detecting radiation-induced genetic damage and supporting their utility in biomonitoring and radiobiological risk assessment.

In this study, monosomies were the most frequent NCAs detected after low-dose XR exposure, likely reflecting radiation-induced disruption of mitotic spindle function and cell cycle control, processes sensitive to oxidative stress (Bakhoum et al., 2015). Such alterations can lead to chromosome missegregation and persistent chromosomal instability across cell generations (Little, 2000). Chromosomes 8 and 21 were most frequently affected, appearing in 50% of irradiated samples, which suggests intrinsic chromosomal vulnerability (Bonassi et al., 2006; Mitelman et al., 2007). These findings are consistent with radiation-induced genomic instability, characterized by the persistence and accumulation of chromosomal alterations over time (Bonassi et al., 2006).

A markedly higher number of SCAs was detected in irradiated samples (28) compared with non-irradiated controls (5), underscoring the high radiosensitivity of human lymphocytes even at low XR doses. The predominant SCAs including: deletions, inversions, isochromosomes, and ring chromosomes, are consistent with radiation-induced DNA double-strand breaks (DSBs), one of the most deleterious consequences of radiation-induced damage. SCAs mainly involved chromosomes 11, 16, and 17, which contain regions enriched in genes related to cell cycle regulation and DNA damage response, potentially increasing their susceptibility to misrepair following irradiation (Goodarzi and Jeggo, 2013; Obe et al., 2002). Recurrent deletions: del(15)(q22) and del(16)(q22) further support the presence of chromosomal regions prone to radiation-induced breakage, possibly influenced by chromatin organization and replication timing (Wang et al., 2020).

In addition, the high frequency of the heterochromatic variant chtb(9)(q12) underscores the sensitivity of constitutive heterochromatin to radiation-induced damage. Pericentromeric regions such as 9q12, enriched in repetitive DNA, are particularly prone to fragility under replication stress and impaired DNA repair, a vulnerability that may be exacerbated by ionizing radiation (Brown and Freudenreich, 2021). These findings support the notion that radiation-induced chromosomal damage is shaped by chromosome-specific features and chromatin architecture rather than being randomly distributed across the genome (Obe et al., 2002; Bickmore and van Steensel, 2013).

Moreover, low-dose XR exposure induced a marked rise in chromosomal fragilities in peripheral blood lymphocytes, which are recognized as sensitive biomarkers of early radiation-induced DNA damage (Amundson et al., 2001). Notably, fra(9)(q12), a rare heritable fragile site associated with specific DNA sequence features, was the most prevalent site, detected in 83.3% of irradiated samples, highlighting its particular sensitivity to low-dose XR (Feng and Chakraborty, 2017). Unlike common fragile sites, which are broadly present and primarily induced by replication stress, fra(9)(q12) reflects site-specific genomic vulnerability that may be exacerbated by ionizing radiation. These findings are consistent with evidence that low-dose radiation can produce subtle yet cumulative genotoxic effects with potential long-term carcinogenic implications (Mothersill and Seymour, 2014).

The markedly lower frequency of chromosomal fragilities, NCAs and SCAs in non-irradiated samples, suggest that XR exposure acts as a direct inducer of chromosomal damage under the experimental conditions evaluated. This evidence underscores the biological relevance of even minimal radiation exposure, as repeated low-dose events in clinical or occupational contexts, may exert cumulative genotoxic effects with potential long-term health implications.

Dicentric chromosomes, although highly specific biomarkers of ionizing radiation–induced damage, were not detected in this study. This is consistent with their intrinsic instability and strong negative selection during mitosis, which limits their persistence at metaphase, particularly at low radiation doses where their expected frequency approaches background levels (∼1/1,000 cells) (Wilkins et al., 2008; Lee Y. et al., 2019). Reliable dicentric detection typically requires first-division metaphases obtained from 48-h cultures with cell-cycle discrimination, conditions not applied here, as lymphocytes were cultured for 72 h without BrdU to enable the characterization of both stable and unstable chromosomal alterations (Lee Y. et al., 2019). Under these conditions, dicentrics are likely eliminated across divisions or repaired through pathways such as non-homologous end joining or homologous recombination (Murmann-Konda et al., 2021). Accordingly, their absence aligns with both the low-dose exposure and the experimental design. To address this limitation, the MN assay was included as a complementary endpoint, allowing detection of unstable chromosomal damage that may not persist to metaphase.

Consistent with the chromosomal alterations observed in irradiated samples, MN frequency was significantly increased compared with non-irradiated controls, underscoring the sensitivity of the MN assay for detecting radiation-induced genomic damage at low doses. This finding confirms that ionizing radiation induces measurable genetic damage, in line with previous evidence showing that radiation promotes DNA strand breaks, acentric fragment formation, and subsequent MN generation during mitosis (Durante and Formenti, 2018). As such, MN formation represents a robust indicator of radiation-induced genomic instability and impaired DNA repair (Fenech, 2000; Holland et al., 2008; Wilhelm et al., 2020).

Additionally, MN frequency was significantly influenced by sex, with women exhibiting higher levels than men regardless of irradiation status. This difference may reflect hormonal influences, particularly estrogen-mediated effects on redox balance and genomic stability, as well as sex-related differences in oxidative damage repair capacity (Schmitz-Feuerhake et al., 2016; Topic et al., 2016; Narendran et al., 2019). The sex-specific increase in MN frequency after irradiation was not accompanied by a parallel rise in metaphase-detected chromosomal alterations, highlighting the distinct sensitivities of these cytogenetic endpoints. While metaphase analysis identifies alterations that persist through cell-cycle progression, the MN assay captures both chromosome breakage and whole-chromosome loss occurring during previous mitotic events, thereby reflecting earlier or transient genomic instability (Fenech, 2007; Norppa and Falck, 2003). Conversely, more stringent cell-cycle checkpoint control and more efficient elimination of heavily damaged cells in female lymphocytes may limit the progression of cells harboring complex aberrations to mitosis, resulting in lower detectable chromosomal alteration frequencies despite elevated MN levels (Rall-Scharpf et al., 2021; Broestl and Rubin, 2021). Collectively, these findings highlight the complementary nature of MN and chromosomal alteration assays and underscore the importance of considering sex as a key biological variable in radiobiological research and radiation risk assessment (Ji et al., 2019; ICRP, 2017).

Cytogenetic analyses, particularly the MN assay, offer a sensitive and reliable approach for detecting early radiation-induced genetic alterations and serve as biomarkers of both, exposure and effect (Fenech, 1993). These tools are especially valuable for occupational and environmental monitoring, including healthcare workers, chronically exposed professionals, patients undergoing radiotherapy, and populations living near radiation sources (Gnanasekaran, 2021; IAEA, 2014; Monteiro Gil et al., 2020). Early detection of cytogenetic damage supports preventive interventions, improves disease risk estimation, and informs evidence-based public health strategies.

Although the adverse health effects of high-dose radiation are well established and underpin current ICRP-based protection frameworks (Stewart et al., 2012), the biological consequences of chronic low-dose occupational exposure remain insufficiently characterized, particularly using genetic damage biomarkers (Mbutu-Austin et al., 2025). In this context, our findings demonstrate that low-dose XR exposure induces detectable chromosomal alterations, fragile sites, and MN formation in human lymphocytes, reinforcing the sensitivity of cytogenetic endpoints for monitoring radiation-induced effects.

Collectively, these results support the integration of cytogenetic biomarkers into radiation protection and biomonitoring strategies, especially for chronically or occupationally exposed individuals. Chromosomal alterations and MN formation are established indicators of genomic instability and have been associated with increased disease risk, including cancer, as supported by epidemiological evidence from radiation workers and atomic bomb survivors (Piotrowski et al., 2017). The detection of chromosomal damage at low XR doses further indicates that genome integrity may be compromised even at exposure levels traditionally considered low. Although adaptive responses such as inducible DNA repair have been described in lymphocytes (Kelsey et al., 1991), these mechanisms do not preclude initial damage induction, highlighting the complexity of low-dose radiation responses and the need to better understand how damage, repair capacity, and individual susceptibility interact to inform radioprotection frameworks.

Low-dose XR exposure was found to induce chromosomal alterations and increase MN formation in human lymphocytes, confirming the high sensitivity of these cells to ionizing radiation. Importantly, the detailed characterization of the type and frequency of chromosomal alterations, encompassing both clonal and non-clonal events, provides refined evidence of how low-dose exposure compromises genome integrity. By delineating specific cytogenetic signatures associated with low-dose XR exposure scenarios, this work fills a critical gap in understanding the biological impact of such exposures and contributes essential data for strengthening radiobiological risk assessments and radioprotection frameworks. Furthermore, sex-related differences in MN frequency indicate that intrinsic biological factors may modulate susceptibility to radiation-induced genetic damage, underscoring the complexity of cellular responses to genotoxic stress. Overall, the findings highlight the value of cytogenetic assays as sensitive tools for biomonitoring radiation effects and support their potential to inform risk assessment, guide preventive strategies, and contribute to public health efforts aimed at minimizing long-term radiation-related health risks.