INTRODUCTION

The majority of the cellular processes that provide for the genetic homeostasis of the body, as well as the reliability of storage and transmission of hereditary information after exposure, are genetically determined. When studying the response of cells to the effect of low doses of ionizing radiation (mainly with a low dose rate), there is a change in the expression of a number of genes, including transcription factors involved in signaling pathways aimed at regulating the functions of lymphocyte subpopulations and the secretion of cytokines and chemokines, which allows exposed cells and tissues to restore genetic homeostasis (Amundson et al., 2003). Thus, low doses of irradiation promote changes in the transcription level of key proteins (p105, JNK1, p38 MAPK, Lck, and ZAP70) involved in signal transduction of CD4+ lymphocytes and changes in the expression of genes that regulate the immune response (Rizvi et al., 2011).

Some changes in immunity are described in the exposed residents of the Techa riverside settlements in the Chelyabinsk and Kurgan regions during the development of long-term medical consequences. This category of people has a reduced number of leukocytes in the blood due to a decrease in the content of neutrophils and lymphocytes, reduced intensity of intracellular oxygen-dependent metabolism of monocytes, activation of lysosomal activity of neutrophils, and inflammatory changes in the cytokine spectrum of blood serum, in particular, a decrease in the content of IL-4 and an increase in the levels of TNFα and IFNγ (Akleev, 2020).

In some cases, a change in the gene expression pattern is involved in the pathogenesis of long-term radiation effects, such as malignant neoplasms and changes in the immune status in exposed people (Shulenina et al., 2012). It has been shown that long-term chronic exposure at low doses leads to a modification of the expression of the transcription factor NFKB1, accompanied by an increase in the expression of IL-1 and IL-6 (Hosoi et al., 2001). An increased mRNA content of the NFKB family genes (NFκB1, NF-κB2, and Rel), as well as genes regulating the proliferation and differentiation of immunocompetent cells, was observed in the long term (after more than 20 years) in individuals with prostate cancer (Savli et al., 2008), thyroid cancer (Cine et al., 2012), and leukemia (Savli et al., 2012), exposed as a result of the Chernobyl accident. The work of (Albanese et al., 2007) reports changes in the activity of 116 cytokine genes and repair after 11–12 years in people exposed to cumulative doses ranging from 0.18 to 49.0 mGy. A detailed description of the picture of the obtained changes indicated a shift in the immune balance towards inflammatory reactions in this category of people in the long term.

Since radiation-induced changes in the system of cellular homeostasis can persist for a long time after exposure, the aim of this work was to evaluate the expression of transcription factors involved in the regulation of systemic immunity in chronically exposed residents of the Techa riverside settlements during the development of long-term somatic-stochastic effects of radiation exposure.

MATERIALS AND METHODS

The samples of peripheral blood from 264 residents of the Techa riverside settlements, who were chronically exposed due to discharges of liquid radioactive waste (LRW) from the Mayak Production Association, were the object of this study. Internal exposure was formed due to radionuclides that entered the body with river water and locally produced products, while external γ-radiation was formed due to contamination of bottom sediments and floodplain soils with radionuclides. The doses to RBM, thymus, and peripheral lymphoid organs were the main dosimetric values that determine the measures of the impact of IR on the body of the examined people.

Massive discharges of radioactive waste began in 1950, and short-lived radionuclides were the main sources of exposure in the first years. Then, the dose rate of external exposure, as well as internal exposure of soft tissues, significantly decreased as a result of protective measures and the decay of short-lived radionuclides; after 1960, they did not exceed 10–5 Gy/year for everyone who lived in riverside areas. The red bone marrow had a slightly different exposure pattern, since the main contribution to the dose formation was made by the long-lived bone-seeking 90Sr, which provides chronic exposure with a monotonically decreasing dose rate, which was less than 10–5 Gy/year for all those exposed by 1985 (Degteva et al., 2019).

Peeple (a) in the period of acute or exacerbation of chronic inflammatory diseases, (b) having oncological and autoimmune diseases, (c) taking antibiotics or hormonal and cytostatic drugs during the examination, or (d) who had contact with genotoxic agents in the course of their professional activities were excluded from this study.

Based on the criteria formulated, two main groups of people examined were identified:

(1) A group of exposed people with individual accumulated doses to RBM over 70 mGy.

(2) The comparison group: people living in similar socio-economic conditions, but with a cumulative radiation dose to RBM not exceeding 70 mGy for the entire period of life (internal control).

The expression of transcription factors in exposed people was studied in separate periods starting from the onset of chronic radiation exposure (after more than 60 years). The group of chronically exposed people consisted of 168 people; the comparison group included 96 people. Table 1 shows the characteristics of the examined groups.

Table 1. Characteristics of the studied groups
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The mean age was 71.04 ± 0.43 years (range 60–87 years) for chronically exposed individuals and 66.45 ± 0.70 years (range 60–87 years) for the comparison group. The vast majority of blood samples in the two groups were obtained from women. The surveyed groups consisted of individuals belonging to two ethnic groups: Slavs (mainly Russians, and also Ukrainians and Belarusians) and Turks (mainly Tatars and Bashkirs).

The mean cumulative dose to RBM in chronically exposed people was 706.3 ± 46.3 mGy (range: 77.8–3507.1 mGy). The mean cumulative dose to the thymus and peripheral lymphoid organs was 131.3 ± 10.0 mGy (range: 2.3–719.2 mGy). The maximum value of the RBM exposure dose rate was obtained by people during the period of increased radiation exposure (1950–1951). Mean dose rate to RBM during the period of maximum exposure in the group of chronically exposed people was 131.3 ± 10.0 mGy/year (range 2.3–719.2 mGy/year), mean dose rate to the thymus and peripheral lymphoid organs during the period of maximum exposure was 38.5 ± 3.8 mGy/year (range 0.3–321.0 mGy/year). All patients signed a voluntary informed consent for the study, approved by the ethics committee of the Urals Research Center for Radiation Medicine, Federal Medical–Biological Agency of Russia.

The material for the study included peripheral blood samples collected in sterile vacuum tubes Tempus Blood RNA Collection Tubes (Applied Biosystem, United States) containing a transport medium for RNA stabilization in bioassays. Blood was taken from the cubital vein in the morning on an empty stomach.

RNA isolation was performed on columns using the GeneJET Stabilized and Fresh Whole Blood RNA Kit (Thermo Scientific™, United States) according to the standard method. Information on the concentration and purity of isolated RNA samples was obtained using a NanoDrop 2000C spectrophotometer (Thermo Scientific, United States). The ratio of optical densities measured at A260/280 for RNA isolated from all blood samples was 2.0 ± 0.05. The initial amount for analysis was 100 ng/µL of RNA of each sample. The reverse transcription reaction for cDNA synthesis was performed using a commercial High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem, United States). The relative quantitative content of mRNA was calculated by real-time PCR using a CFX96 Touch amplifier (Bio-Rad Laboratories, United States).

Expression of the analyzed genes was quantified using the 2–ΔΔСt (Livak et al., 2001). Data were evaluated relative to the mRNA levels of the housekeeping genes COMT and B2M. Amplification curves were analyzed in the Bio-Rad CFX Manager 2.1 program (Bio-Rar Laboratories, United States) using the threshold line method. The calculation was carried out taking into account three repetitions for each gene and the amplification efficiency, which was obtained by constructing the calibration curves. We used commercial primer/probe kits from Applied Biosystems, United States: STAT3 (Hs00374280_m1), GATA3 (Hs00231122_m1), MAPK8 (Hs01548508_m1), NFKB1 (Hs00765730_m1), FOXP3 (Hs01085834_m1), and TBX21 (Hs00894392_m1).

Statistical analysis of the study data was performed using standard methods of mathematical and statistical processing using software packages for applied statistical analysis (Statistica v. 10.0 and SigmaPlot). The Mann–Whitney U-test was used to assess intergroup differences in trait values. The type of dependence of the expression of transcription factors on the dose characteristics in exposed people was calculated using correlation and regression analysis. For all criteria and tests, differences were considered statistically significant at p < 0.05. At 0.05 < p < 0.1, the difference was considered as a trend towards a significant difference and the need to increase the number of observations.

RESULTS

The results of determining the expression of mRNA of the immunoregulatory genes GATA3, FOXP3, TBX21, STAT3, NFKB1, and MAPK8 in the peripheral blood cells of the examined groups are shown in Fig. 1.

Fig. 1.
figure 1

Expression of the GATA3, FOXP3, TBX21, STAT3, NFKB1, and MAPK8 genes in the comparison group and the group of chronically exposed people (mean and error of the mean). # Significant differences in gene expression between the comparison group and chronically exposed individuals.

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It is noteworthy that the expression of the transcription factors MAPK8 (1.51 ± 0.26 rel. units versus 1.12 ± 0.10 rel. units, at p = 0.01) and NFKB1 (1.10 ± 0.07 rel. units versus 0.91 ± 0.04 rel. units, at p = 0.02) was statistically significantly reduced in the group of chronically exposed individuals, relative to the comparison group units.

In addition, there was a trend towards an increase in the relative content of mRNA of the FOXP3 (5.43 ± 3.95 rel. units versus 10.01 ± 2.94 rel. units) and TBX21 (3.21 ± 1.43 rel. units versus 5.53 ± 1.89 rel. units) genes in the group of chronically exposed people. However, no statistically significant differences were found due to the high degree of data variability and as a result of the large standard error of the mean.

Since the cumulative doses of radiation in chronically exposed people were in a fairly wide range of values, it was of interest to evaluate the mRNA expression of the studied genes depending on the absorbed dose to RBM. For this purpose, the people examined were conditionally divided into three dose subgroups e (those exposed in the range of intermediate doses, group 1, 77.8–484.4 mGy (80 people), and group 2, 522.6–994.4 mGy (50 people), and those irradiated in the range of high doses, group 3, >1000 mGy (1032.1–3507.1 mGy) (38 people).

The data presented in Fig. 2 demonstrate a statistically significant low relative content of mRNA of the MAPK8 gene in chronically exposed people with intermediate accumulated doses to RBM: 77.8–484.4 mGy (p = 0.08) and 522.2–994.4 mGy (p = 0.03) compared with the comparison group.

Fig. 2.
figure 2

Expression of the GATA3, FOXP3, TBX21, STAT3, NFKB1, and MAPK8 genes in dose subgroups of exposed people (mean and error of mean). # Significant differences in gene expression between the comparison group and chronically exposed individuals (doses to RBM 77.79–484.39 mGy); ## significant differences in gene expression between the comparison group and chronically exposed people (radiation doses to RBM 522.16–994.40 mGy).

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As for the results obtained for the NFKB1 gene, expression of which was reduced in the total sample of chronically exposed individuals, statistically significant differences were obtained only between the comparison group and the subgroup of individuals exposed in the dose range of 77.8–484.4 mGy (p = 0.02).

Thus, differences in the transcriptional activity of the above genes between the compared groups were mainly noted for people whose accumulated doses to RBM were in the range of intermediate values.

According to the data presented in Table 2, there were no statistically significant correlations between the relative content of mRNA of the immunoregulatory genes GATA3, STAT3, MAPK8, NFKB1, FOXP3, and TBX21 in the values of the accumulated doses to RBM, thymus, and peripheral lymphoid organs, as well as in the values of the dose rates to RBM, thymus, and peripheral lymphoid organs during the period of maximum radiation exposure. Nevertheless, a positive relationship was found at the trend level, demonstrating an increase in the expression of the GATA3 gene mRNA with the dose to the RBM (p = 0.06) and the thymus and peripheral lymphoid organs (p = 0.07).

Table 2. Spearman’s rank correlation coefficients (R) between the relative mRNA content of the studied genes and the doses and dose rates in the period of maximum exposure
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At present, the activation of some intracellular signaling cascades involved in the regulation of the immune system after radiation exposure has been proven (Akleev et al., 2019). As part of this work, correlation analysis was used to evaluate the relationship between the transcriptional activity of the studied genes in chronically exposed people and in the comparison group.

The results presented in the form of correlation matrices (Figs. 3, 4) indicate the presence of weak positive relationships in two samples between the expression of mRNA of the SATA3 and GATA3 genes (in the comparison group (R = 0.28, p = 0.005) and in the group of chronically exposed people (R = 0.17, p = 0.02)), and mRNA expression of the GATA3 and NFKB1 genes (in the control group (R = 0.27, p = 0.008) and in the group of chronically exposed people (R = 0.19, p = 0.01)).

Fig. 3.
figure 3

Matrix of pairwise correlation coefficients of mRNA expression of the studied genes in peripheral blood cells in people constituting the comparison group.

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Fig. 4.
figure 4

Matrix of pairwise correlation coefficients of mRNA expression of the studied genes in peripheral blood cells in chronically exposed people.

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In addition, a strong positive correlation was found in the two studied samples between the mRNA expression of the FOXP3 and TBX21 genes (in the comparison group (R = 0.63, p = 0.0001) and in the group of chronically exposed people (R = 0.78, p = 0.0001)). A moderate positive correlation between FOXP3 and NFKB1 mRNA expression in the comparison group (R = 0.40, p = 0.02) was a distinctive feature of the comparison group. Meanwhile, a weak inverse relationship between the expression of mRNA of the FOXP3 and STAT3 genes (R = –0.26, p = 0.02) and a weak direct relationship between the expression of mRNA of the NFKB1 and MAPK8 genes (R = 0.22, p = 0.02) were found in the group of chronically exposed people.

Thus, the results of this study indicate that chronic radiation exposure with predominant exposure to human RBM can permanently modify the transcriptional activity of the immunoregulatory genes MAPK8 and NFKB1.

DISCUSSION

It is well proven that ionizing radiation is able to modulate the expression of genes, the products of which coordinate the overall cellular response to stress, including the immune response (Fachin et al., 2009; Morandi et al., 2009). In turn, the aberrant activity of homeostatic genes can serve as the primary cause for the development of both early and late effects of exposure.

The expression of mRNA of immunoregulatory genes was carried out in chronically residents of the Techa riverside settlements (Chelyabinsk and Kurgan regions) in the long term. It has been shown that statistically significant shifts in the functional state of the transcriptome are observed in this cohort of individuals even after so many years after the onset of radiation exposure.

The results of our study showed that the residents of the Techa riverside settlements showed a statistically significant decrease in the expression of mRNA of transcription factors NFKB1 and MAPK8 relative to internal control (people whose exposure doses to RBM did not exceed 70 mGy) during the period of development of late effects (malignant neoplasms, leukemia, and diseases of the cardiovascular system) after chronic radiation exposure with predominant exposure to RBM.

Obviously, post-radiation changes in the transcriptional activity of the NFKB1 and MAPK8 genes in chronically exposed individuals can affect the state of immunity and cellular homeostasis.

The NFKB1 gene encodes the transcription factor of the same name, which is a transcriptional regulator of immune response genes. In particular, entering the cell nucleus, this transcription factor modulates the expression of genes encoding antigen receptors on immune cells, cell adhesion molecules, pro-inflammatory cytokines (TNF, IL1, LPS), and chemoattractants (MCP-1) (Cartwright et al., 2016). In addition, NFKB1 controls the expression of a number of genes involved in a wide range of biological functions of the cell. A number of reports state that NFKB1 is able to influence the transcription of genes encoding apoptotic proteins of the BCL-2 family, such as BCL-XL, BFL-1/A1, Nr13, BAX, and BCL-2, thereby blocking the initiation of apoptosis, after acute exposure (Chen et al., 2002). However, the decrease in NFKB1 mRNA expression, which was found in exposed individuals, should in fact lead to the opposite effect.

This fact can indirectly confirm the data of our earlier studies, in which we demonstrate a significant change in the transcriptional activity of the apoptotic BAX and BCL-2 genes, leading to a shift in the balance of cellular homeostasis towards the induction of the apoptosis process in peripheral blood mononuclear cells in exposed people (Nikiforov et al., 2020).

The results obtained once again confirm the idea that the nature of changes at the molecular level directly depends on the nature of exposure and the magnitude of the exposure dose. Thus, low dose rate exposure results in a gene expression pattern that is different from acute radiation exposure.

MAPK8, the expression of which was also significantly reduced in the group of exposed people, is a substrate for many cellular processes, in particular, the proliferation and development of immunocompetent cells. MAPK8 plays a key role in the differentiation of T lymphocytes from T helpers into T helper 1 (Th1) (Dent et al., 2003). This transcription factor also plays an important role in the process of apoptosis and promotes its active initiation in stressed cells due to phosphorylation of key regulatory factors, including p53 (Yue et al., 2020).

Not all immunoregulatory genes studied have demonstrated transcriptional responses to chronic radiation exposure. Statistically significant deviations in the expression of genes encoding Stat3 (a factor in the growth and development of immunocompetent cells), Gata3 (a factor in inflammatory and humoral immune responses, a regulator of the development of the T-cell link), Foxp3 (a regulator of the development and functioning of regulatory T-cells), and Tbx21 (factor activating the proliferation of T-helper type 1) proteins in chronically exposed people in relation to the comparison group were not detected.

However, there were tendencies towards an increase in the expression of mRNA of the TBX21 and FOXP3 genes in the group of chronically exposed people, and a positive correlation between the expression of the GATA3 gene mRNA and the radiation doses to the RBM, thymus, and peripheral lymphoid organs. These data are preliminary; it is necessary to continue this study with expanded samples of the people examined, in order to clarify the significance of the above factors in the development of late effects of radiation.

Depending on radiation factors, such as dose and dose-rate, certain signaling cascades are subject to change, which can modify the functions of cells of the immune system in humans (Azimian et al., 2015). However, to date, the signaling pathways involved in the immune response to chronic low dose-rate radiation exposure remain poorly understood.

Data on the study of cooperative interactions of genes are of interest. In addition to the relationships that were recorded in the two samples studied, there were correlations that were typical only for the group of exposed people. In particular, there is a positive weak correlation between the expression of the previously mentioned mRNAs of the MAPK8 and NFKB1 genes.

As mentioned earlier, the products of these genes regulate the immune response through the control of the processes of proliferation and differentiation of immunocompetent cells. According to the published data, these genes are key activators of signaling pathways involved in inflammatory responses (Loza et al., 2007). In addition to the production of pro-inflammatory cytokines (TNF-a, IL-6, etc.), these transcription factors control the expression of chemoattractant genes in macrophages, neutrophils, and T cells (Dent et al., 2003). However, the involvement of MAPK8 and NFKB1 in the pathogenesis of inflammatory reactions in exposed people in the long term requires further study.

Thus, chronically exposed residents of the Techa riverside settlements with predominant exposure of the red marrow (the maximum absorbed doses reached 3507.1 mGy) in the long term showed features of the expression of transcription factors MAPK8 and NFKB1 involved in the regulation of human systemic immunity. Taking into account the importance of the expression of these transcription factors in maintaining the genetic homeostasis of immunocompetent cells, it can be assumed that changes in the regulation of systemic immunity in exposed people in the long term can be mediated by modification of their transcriptional activity.