According to current understanding, telomeres are essential terminal regions of chromosomes that perform a multitude of functions, with the primary one being the maintenance of genomic integrity. It has been noted that telomere length varies widely not only among individuals but even within a single cell and across chromosomal pairs, which can be explained by uneven telomere shortening [1][2]. This phenomenon of telomere length distribution along the arms of individual chromosomes is referred to as the “chromosomal telomere profile” [3]. The telomere profile can serve as a potential biomarker for predicting the risk of certain diseases and for assessing the impact of adverse external factors, including ionizing radiation [4]. Consequently, research aimed at studying the influence of radiation exposure on telomere length is highly relevant. Exposure of individuals due to atomic bombings in Hiroshima and Nagasaki to doses conventionally categorized as low (5–700 mSv) and high (over 700 mSv) led to telomere shortening; these alterations persisted even in the long term (50–68 years post-exposure) in the high-dose group [5]. Conversely, some studies have reported an increase in telomere length in exposed individuals, which has been associated with elevated telomerase expression [6].

An assessment of relative telomere length in nuclear industry workers exposed to internal alpha (0.05 Gy; 0.05–0.1 Gy; > 0.1 Gy) and external gamma (1 Gy; 0.1–1.5 Gy; > 1.5 Gy) radiation found this parameter to decrease only after low-dose exposure, for both external gamma radiation (at doses < 1 Gy) and internal alpha radiation (at doses < 0.05–0.1 Gy). At higher doses, this parameter did not differ from the values in the non-exposed control group [7]. A study involving individuals chronically exposed at a wide dose range (0–4.7 Gy) revealed a decrease in relative telomere length under chronic exposure (0.6–4.7 Gy) compared to the control group for specific chromosomal arms of meta- and acrocentric chromosomes. No monotonic decrease in telomere length was observed for chromosomes within a single cell or across different arms of the same chromosome [1]. This indicates that telomere length is a dynamic structure determined not merely by chronological age but rather by the combined influence of numerous endogenous and exogenous factors.

At present, researchers are increasingly addressing the relationship between telomere length and cell and body radiosensitivity [8]. For instance, certain diseases characterized by clinical hyper-radiosensitivity (ataxia-telangiectasia, Fragile X syndrome, Fanconi anemia) were shown to be associated with impairments in telomere maintenance [9]. Investigations into the radiosensitivity of human fibroblasts exposed to X-ray radiation in vitro demonstrated that cell clones that retained viability after a 4 Gy irradiation dose exhibited increased telomere length compared to non-irradiated clones. The authors advanced a hypothesis that cells with longer telomeres are more radioresistant, and that radiosensitivity correlates with telomere loss rather than with their average length [4].

A number of studies have shown that the quantity of ultra-short telomeres, rather than their average length, serves as the most informative predictive marker for the development of certain diseases [10]. For instance, an association between the frequency of ultra-short telomeres and various human pathologies, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis, has been established [11].

All existing methods for studying chromosome telomeres can be broadly distinguished into two groups: molecular and molecular-cytogenetic. The molecular-cytogenetic method of quantitative fluorescence in-situ hybridization (Q-FISH) on metaphase chromosomes is the most suitable approach for the individual assessment of telomeric regions. This method employs fluorescently labeled peptide nucleic acids (PNA), which specifically hybridize with denatured telomeric DNA. Using the Q-FISH method, telomeric regions can be examined in each chromosome in every cell, since chromosome karyotyping is performed during the analysis [1].

In this study, we focuse on telomere length in individuals chronically exposed to a wide dose range due to residence in radionuclide-contaminated areas, during long-term follow-up (60–65 years post-exposure). The radiation exposure was combined: internal, i.e., due to the intake and accumulation of 89,90Sr radionuclides in the body, and external — from γ-radiation. The critical organ for radiation exposure was the red bone marrow (RBM). Doses to the RBM were predominantly accumulated before 1960 [12].

Our aim was to investigate telomere length using the Q-FISH method in exposed individuals and to identify the proportion of ultra-short and ultra-long chromosomal telomeric regions in this cohort.

The study involved 43 female donors from different age groups (21–28; 60–67; 71–83 years). The inclusion criteria were the absence of a history of autoimmune or oncological diseases, and chronic inflammatory diseases in the acute phase. Individuals taking cytostatics or antibiotics were excluded. Health status information for the exposed individuals was obtained from the Database “Chelovek” (Man); individual doses were calculated using TRDS-2016 in the Biophysics Laboratory. Information on cancer history in the examined individuals was provided by an Epidemiology Laboratory [13].

At the first stage, an investigation of the age-dependence of relative telomere length was conducted in a control group (donor characteristics are presented in Table 1). Three age groups of donors were formed:

Although donors from the older age group resided in radionuclide-contaminated areas, their accumulated bone marrow dose did not exceed 0.05 Gy over their lifetime. Consequently, hereafter (in stages II and III of the study), these donors will be referred to as non-exposed individuals or the comparison group.

At stage II, the reference for average telomere length was established in the comparison group. At stage III, taking into account the established reference values, telomere length was studied in exposed individuals, including analysis based on age and bone marrow dose.

With the purpose of assessing the influence of ionizing radiation on telomere length, two groups of exposed donors were formed: those exposed to doses ranging 0.14–0.86 Gy (10 individuals aged 69–80 years) constituted the medium-dose exposure group; the high-dose exposure group comprised 12 individuals aged 71–84 years with bone marrow doses of 1.04–3.1 Gy (Table 2).

Furthermore, a case-control analysis was conducted to eliminate the influence of the age factor. To that end, potential controls for each donor (“case”) from the medium-dose exposure group were selected based on the criterion of matching the age at the time of examination. The bone marrow dose was considered the influencing factor. Four pairs of donors of similar age were formed. In each pair, one donor had an established accumulated bone marrow dose ranging 0.61–0.86 Gy, while the other donor in the pair either had not been accidentally exposed or had a dose of ≤ 0.05 Gy (Table 3).

For the case-control study within the high bone marrow dose range, five pairs of donors of similar age were selected. One donor in each pair had a dose exceeding 1 Gy, and the other donor either had not been accidentally exposed or had a dose of ≤ 0.05 Gy (Table 3).

The subject of the study was the nuclear chromatin of T-lymphocytes from peripheral blood stimulated with phytohaemagglutinin (PHA). T-lymphocytes were chosen due to the ease of obtaining the source material and their sufficiently high concentration. T-lymphocytes do not divide in peripheral blood and reside in the G0 phase of the cell cycle, representing a naturally synchronized cell population in the body. Lymphocyte precursor cells are irradiated directly in the RBM.

Blood was drawn into vacuum plastic blood collection tubes containing lithium heparin.

The culture mixture was prepared from the following components: 2 mL of whole heparinized blood, 5 mL of RPMI-1640 medium (PanEco, Russia), 0.5 mL of selected fetal bovine serum (PAA Laboratories, Austria). PHA was added to a final concentration of 20 μg/mL (PanEco, Russia). Cells were cultured in a CO2 incubator (SANYO MCO-18AIC, Japan) at 37.5 °C for 54 h. Three hours prior to the end of cultivation, colchicine was added to achieve the final concentration of 0.1 μg/mL (PanEco, Russia).

Subsequently, metaphase cells underwent hypotonic treatment, fixation of metaphase plates (using a 3:1 mixture of 95% medical-grade ethanol and glacial acetic acid), and chromosome slide preparation. For fluorescent staining using the Q-FISH method, slides were treated with RNAse (100 ng/mL) and pepsin solutions, followed by denaturation of the probe and specimen DNA. Hybridization was performed in accordance with the manufacturer’s protocol, which was supplied with the original reagent solutions.

To assess the length of chromosomal telomeric regions, DAKO probes (Denmark) were used. Centromeric probes (Metasystems; Germany) were applied to stain the centromeric region of chromosome 2, which served as a reference signal. Analysis of fluorescently stained specimens was performed using an AxioImager Z2 fluorescence microscope (Zeiss; Germany) equipped with DAPI and SpO (spectrum orange) filters.

The intensity of the recorded fluorescent signal was measured using the Isis specialized image analysis software. For each subject, 20–30 cells were analyzed. Results were obtained separately for the short (p-) and long (q-) arms of each of the 46 chromosomes. Telomere length was relative, being expressed as a percentage of the telomeric (T) signal length relative to the centromeric (C) signal length — (T/C%). The method for assessing telomeric region length is described in detail in [14].

Standard methods of descriptive statistics were used to process the data obtained, including calculation of the median, its 95% confidence interval (95% CI), and the 5th and 95th percentiles (%). Confidence intervals were calculated using a bootstrap procedure with n = 1000 resamples. Intergroup comparisons were performed using the non-parametric Mann–Whitney U test.

The threshold for ultra-short telomeres was defined as the value below which the lowest 5% of all telomere length measurements in the non-exposed group fall. Similarly, the reference value for ultra-long telomeres was calculated as all values exceeding the 95th percentile of the measurement distribution. The obtained reference values were used for comparative analysis with data on the relative telomere length in individuals exposed at various dose ranges.

When comparing the frequency of ultra-short and ultra-long telomeres in the case-control study and against the reference value, the χ² test was used. Multivariate analysis was performed to determine the association of telomere length with age and dose. Spearman’s correlation coefficient and regression analysis were employed to assess the relationship between the dose and the number of ultra-short and ultra-long telomeres per individual. Statistical analysis was conducted using the following software packages: Past 4.01 (Oyvind Hammer; UK), Statistica 10 (StatSoft, USA), and SigmaPlot (SYSTAT Software, USA).

During the study, data on telomere length measurements were obtained for three age groups of non-exposed donors (Table 4).

In the donor group aged 20–28 years, 4331 measurements of telomeric regions were performed. The minimum recorded telomere length was 0%, the maximum was 900.0%, and the median telomere length was 31.0%.

Simultaneously, in the middle-aged donor group (60–67 years), 22,311 measurements of telomeric regions were performed. The telomere length range (min–max) was 0–967.9%, and the median value was 13.0%. In the donor group aged 71–83 years, 9732 measurements were obtained. The minimum telomere length was 0%, the maximum was 141.2%, and the median was 5.8%.

The values for ultra-short telomeres in the younger donor group ranged 0–6.3%; for donors aged 60–67 and  71–83 years the values were 0–1.8 and 0–0.7%, respectively. Meanwhile, the ranges for ultra-long telomeres were 221.2–900.0% for donors aged 20–28 years, 105.9–967.9% for donors aged 60–67 years, and 25.6–141.2% for donors aged 71–83 years.

It was established that median telomere length decreased with an increase in donor age. Pairwise comparison of values in the studied control groups revealed statistically significant differences (p = 0.0001). Regression analysis demonstrated a statistically significant (p < 0.001; R² = 0.07), albeit extremely weak, linear dependence between relative telomere length and donor age:

y = 86.1 – 26.7 x, (1)

y — relative telomere length, х — age of the examined individuals.

For the subsequent analysis of telomere length in exposed individuals, a reference value of 0.7% was adopted for ultra-short telomeres, corresponding to the lower bound of the 90% telomere length range in non-exposed donors aged 71–83 years. Ultra-long telomeres were defined using the upper bound of the 90% telomere length range — 25.6%.

Table 5 presents the telomere length measurement data for exposed donors. The comparison group consists of non-exposed donors aged 71–83 years.

It can be seen that the median telomere length for all exposed individuals was 10.3%, which is statistically significantly higher than that in the comparison group (5.8%; p = 0.0001). At the same time, the 90% interval of telomere length ranged 1.4–56.5%.

Considering the reference value for ultra-short telomeres, within the group of exposed donors (0.14–3.1 Gy), 616 telomere measurements were classified as ultra-short, constituting 1.6% of all measurements. Within the reference range for ultra-long telomeres, exposed donors had 7491 measurements, accounting for 19.5%. Thus, exposed individuals exhibited a lower number of ultra-short telomeres (χ² = 397.03; p < 0.0001) and a higher number of ultra-long telomeres (χ² = 1184; p < 0.0001) relative to the comparison group.

In the medium-dose exposure group (0.14–0.86 Gy), the median telomere length was 11.9% (min–max: 0–643.4%). For donors exposed to high doses, the median was 10.6% (min–max: 0–332.1%), which is statistically significantly lower than in the medium-dose group (p = 0.0001).

Relative to the reference values, the frequency of ultra-short telomeres in donors exposed to medium doses was 1.3%, and 1.4% in those exposed to high doses The frequency of ultra-long telomeres was 24% in the medium-dose group and 19.5% in the high-dose group, which was statistically significantly different from the values in the comparison group (p = 0.0001). Thus, in exposed individuals with a high accumulated bone marrow dose, the frequency of ultra-short telomeres is similar to that in the medium-dose group. At the same time, in the former group, the number of ultra-long telomeres is higher.

The relationship between telomere length, age, and bone marrow dose was established by a two-factor analysis (general linear model). A statistically non-significant model where telomere length in the entire sample of exposed individuals did not depend on age or bone marrow dose was obtained.

Table 6 presents data on the pairwise comparison for the case-control analysis, where the “cases” are individuals with medium (0.14–0.86 Gy) and high (1.04–3.1 Gy) accumulated bone marrow doses.

In all examined pairs from the medium dose group, a statistically significant increase in telomere length was detected in exposed donors compared to their controls.

In the group of individuals exposed to high doses, a statistically significant increase in telomere length in exposed donors compared to their controls in four out of five pairs was found. In Pair No. 1, a statistically significant decrease in telomere length was observed in the exposed donor relative to their control. However, in this specific case-control pair, the frequency of ultra-short telomeres did not differ from the control values (0.8% vs. 0.7%; χ² = 0.03; p = 0.86), while the frequency of ultra-long telomeres was significantly lower (1.4% vs. 2.9%; χ² = 8; p = 0.005) (Table 7).

Table 7 presents the frequencies of ultra-short and ultra-long telomeres for each case-control pair (based on reference values) among individuals with medium and high bone marrow doses.

The study yielded statistically significant data on changes in the frequency of ultra-short telomeres in three out of four pairs (p < 0.0001), with the exception of Pair No. 3 (p = 0.19). At the same time, in all examined pairs, the frequency of occurrence of ultra-short telomeres in exposed donors was statistically significantly lower than that in the controls. Furthermore, the frequency of ultra-long telomeres in all pairs of donors exposed to doses of 0.14–0.86 Gy was statistically significantly higher compared to the control group (p < 0.0001).

It can be seen that for individuals exposed in the dose range of 1.04–3.1 Gy, the frequency of ultra-short telomeres is statistically significantly lower relative to the control in four out of five pairs. No statistically significant differences in the frequency of ultra-short telomeres were observed in Pair No. 1. Also, in this same pair, the exposed donor had a lower frequency of ultra-long telomeres compared to the control. In the remaining four examined pairs, an increase in the frequency of ultra-long telomeres was observed in exposed individuals relative to the controls.

Subsequently, for the donors selected into the case-control study, Spearman’s rank correlation coefficient was used to assess the relationship between the proportion of ultra-short telomeres and the dose. The results found no correlation between ultra-long telomeres and bone marrow dose; however, a trend towards a decrease in the frequency of ultra-short telomeres with an increase in bone marrow dose was observed. A statistically significant inverse correlation of moderate strength was established between the frequency of ultra-short and ultra-long telomeres in exposed donors (rs = – 0.654, p = 0.004).

Subsequently, the most suitable regression model describing the dependence of ultra-short telomere frequency on bone marrow dose was fitted (Figure).

The Figure shows that an increase in dose is associated with an exponential decrease in the number of ultra-short telomeres.

y = 4.3 e–1.5D [ R² = 0.23, p = 0.0036], (2)

y — frequency of ultra-short telomeres, D — bone marrow dose, Gy.

Thus, it was demonstrated that exposed individuals exhibit a reduced frequency of ultra-short telomeres and an increased frequency of ultra-long telomeres relative to the comparison group. An exponential decrease in the frequency of ultra-short telomeres with an increase in bone marrow dose was observed.

The present study continues a project investigating the effects of chronic radiation exposure on chromosomal telomeric regions. As part of this long-term project, the length of telomeric regions in individual chromosome pairs in chronically exposed individuals was examined, and the frequency of inversions involving telomeric regions and the frequency of telomeric region loss in metaphase chromosomes from cultured peripheral blood T cells of exposed residents in the Southern Urals were assessed [14]. To that end, a method of fluorescent staining of chromosomal telomeric regions was proposed and validated. The use of the Q-FISH method is justified by its capacity to investigate telomeric regions in each chromosome within a donor’s cells. This enables the characterization of the telomere profile.

The present study is the first of its kind to conduct an analysis of the influence of both radiation and non-radiation factors (age) on the frequency of ultra-short and ultra-long telomeres. The frequency of ultra-short and ultra-long telomeres in exposed individuals was assessed based on reference values for telomeric region length, which were calculated for a comparison group of a similar age range. The reference range for telomere length in the comparison group (age 71–83 years) was 0.7–25.6%. Telomeres with a length < 0.7% were classified as ultra-short, while those above 25.6% were classified as ultra-long.

A statistically significant increase in median telomere length was demonstrated in the group of exposed individuals compared to the comparison group. At the same time, wide variability in telomere length was observed in both exposed individuals and the comparison group, which is consistent with previously obtained data [1][15]. This variability may indicate the circulation of T cells with different replicative ages in the peripheral blood.

According to the results obtained, in the overall sample of exposed individuals, the frequency of ultra-short telomeres was lower, and that of ultra-long telomeres was higher relative to the comparison group. These findings were further supported by an assessment using the case-control method with age-matched donors. The presence of longer telomeres in exposed individuals may indicate molecular-genetic features associated with either increased telomerase activity [16] or epigenetic changes in other enzymes regulating telomere replication and repair [17][18], as well as the selection of more radioresistant cells and the death of radiosensitive ones [4]. The donor selection criteria for the cytogenetic study may have also played a role, since the sample inherently comprised the healthiest individuals relative to the general population. This is a necessary selection criterion, since excluded medical conditions, therapeutic procedures, and medications can distort cytogenetic data [1].

Furthermore, a statistically significant decrease in median telomere length was observed in the donor group with a high bone marrow dose compared to the group exposed to medium doses. This difference was primarily associated with a reduced frequency of ultra-long telomeres in the high-dose group, while the frequency of ultra-short telomeres remained at the same level as in individuals with medium doses. At the same time, the frequency of ultra-long telomeres did not depend on the bone marrow dose, whereas the frequency of ultra-short telomeres decreased exponentially with an increase in dose. The presented patterns require further in-depth investigation. Provided that subsequent studies confirm the relationship between the frequency of ultra-short telomeres and radiation dose, this parameter could be used as an indicator of radiation exposure or a biomarker of cell radiosensitivity [19][20].

The present study provides data on the influence of chronic radiation exposure on telomere length in women, obtained using the Q-FISH method. This method enables the determination of relative telomeric region length by calculating the ratio of the telomeric signal fluorescence intensity to the centromeric signal fluorescence intensity of chromosome 2, expressed as a percentage (T/C, %). Ultra-short and ultra-long telomeres in exposed individuals were identified relative to the reference telomere length value calculated in the comparison group.

Our results revealed a statistically significant increase in median telomere length in exposed individuals relative to the comparison group. The increase in telomere length is associated with a decreased frequency of ultra-short telomeres and an increased frequency of ultra-long telomeres in exposed individuals. The study conducted using a case-control design with age-matched donors confirmed that this pattern persists independently of age, underscoring the significance of the radiation factor. The dependence of ultra-short telomere frequency on bone marrow dose is described by a non-linear regression manifested in an exponential decrease in the frequency of ultra-short telomeres with an increase in dose. This phenomenon highlights the importance of further investigation into the role of ultra-short telomeres as a potential marker of radiation exposure.

Authors’ contributions. All authors confirm that their contributions meet the ICMJE criteria for authorship. The primary contributions are distributed as follows: Yana V. Krivoshchapova — research concept and design, conducting laboratory investigations, acquisition of primary data, statistical analysis of results, drafting the article; Yulia R. Akhmadullina — statistical analysis and interpretation of data, writing, editing, and final preparation of the manuscript.