ISSN 00063509, Biophysics, 2011, Vol. 56, No. 2, pp. 333–338. © Pleiades Publishing, Inc., 2011. Original Russian Text © I.I. Pelevina, I.V. Oradovskaya, Yu.G. Mansurova, A.V. Aleshchenko, M.M. Antoshchina, O.V. Kudryashova, E.Yu. Lizunova, M.F. Nikonova, A.N. Osipov, N.I. Ryabchenko, V.V. Feoktistov, A.M. Serebryanyi, 2010, published in Radiatsionnaya Biologiya. Radioekologiya, 2010, Vol. 50, No. 5, pp. 501–507.
RADIOBIOLOGY AND RADIOECOLOGY
The Correlation between Molecular Cellular Parameters and Immune Status in the Blood Lymphocytes of Chernobyl Nuclear Accident Cleanup Workers I. I. Pelevinaa, I. V. Oradovskayab, Yu. G. Mansurovab, A. V. Aleshchenkoc, M. M. Antoshchinad, O. V. Kudryashovaa, E. Yu. Lizunovaa, M. F. Nikonovab, A. N. Osipova, N. I. Ryabchenkod, V. V. Feoktistovb, and A. M. Serebryanyic a
Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow, Russia Email:
[email protected] b Institute of Immunology, Ministry of Health, Moscow, Russia c Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia d Medical Radiological Research Center, Russian Academy of Medical Sciences, Obninsk, Russia Received June 10, 2010
Abstract—Genome damage, namely, the frequency of cells with micronuclei (MN) and chromosome aber rations, the level of DNA doublestrand breaks, and concentration of reactive oxygen species (ROS), as well as 28 immunological characteristics were studied in the blood lymphocytes of Chernobyl nuclear accident cleanup workers. The goal of the work was to examine the cytogenetic and molecular biological changes in the blood lymphocytes of exposed individuals 24 years after the accident and to find the correlations between genome damage and immunity parameters. The rate of cells with MN and the total rate of the cells with all types of chromosome aberrations did not differ from those of unexposed individuals; however, the amount of chromosome type aberrations and DNA doublestrand breaks were elevated in the lymphocytes of cleanup workers, whereas the concentration of reactive oxygen species was decreased. As compared with the control population cohort, cleanup workers displayed a statistically significant increase in the percent of cytotoxic CD8+ T lymphocytes, CD16+ natural killers, CD3+CD16+CD56+ natural killer cells (NKT cells, display ing antiviral and antitumor activities), HLADR+ (a marker of late lymphocyte activation), and regulatory T lymphocytes (CD4+CD25+high). The level of serum immunoglobulin (IgA) increased in a statistically signif icant manner. The index of immune regulation decreased along with the values of phagocytic activities of neutrophils (PAN) and macrophages (PAM). Liquidators displayed statistically significant (p < 0.05) corre lations of the rate of cells with MN with the content of regulatory T lymphocytes (CD4+CD25+high) and between the reactive oxygen species concentration and the content of activated T lymphocytes. A consider ably larger number of correlations was detectable at the level of sufficiently reliable trends at p < 0.10, in par ticular, of the rates of chromosome aberrations and DNA doublestrand breaks with the contents of natural killers and regulatory T lymphocytes, as well as of the rates of the cells containing MN and DNA DSBs with PAN. This suggests that the genomic instability induced by exposure 24 years ago during the cleanup activities after the Chernobyl nuclear accident is manifested now as an increased level of genome damage and a decrease in oxidative status, which can result in the cell and humoral immunity disturbance. Keywords: lymphocytes, cleanup workers, Chernobyl nuclear accident, genome damage, immune status DOI: 10.1134/S0006350911020242
Over the last 24 years, the consequences of the Chernobyl nuclear accident for the health of cleanup workers (participants in the emergency cleanup) are still discussed. In this work, we attempted to compare the parameters of the immune status of cleanup work ers with the immune status of unexposed individuals (the control population cohort from the same region), to detect changes, and to compare these data with the changes in the genome and oxidative status state in the exposed individuals.
The object of the study was the human peripheral blood lymphocytes. These cells are formed of stem cells and after several in vivo transformations convert into functionally mature cells that enter the blood, where they circulate for a certain relatively short period to be replaced with new cells. The changes induced in the stem cells by ionizing radiation are retained for a long time, as is demonstrated by the genomic instability (GI), which appears as an increased rate of the lymphocytes with aberrations, even many years after exposure [1].
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In this work, we used an integrated approach based on concurrent comparative estimation of the cytoge netic and immunological changes initiated many years ago. It is assumed that such an approach will make it possible to assess the roles of oxidative homeostasis, damage to DNA and the chromosome apparatus, and immune status in the formation of GI of lymphocytes and changes in their functional properties. Such approaches have not been so far applied in the world practice for studying the mechanisms of genomic instability. MATERIALS AND METHODS Cytogenetic Examination All reagents were purchased from Paneko (Mos cow, Russia). Metaphase analysis of the rate of the lymphocytes with unstable chromosome aberrations was performed using a standard protocol for the culti vation of peripheral blood lymphocytes [2]. Heparin ized blood (1 ml) was supplemented with 8 ml of the RPMI medium containing 15% fetal bovine serum. Lymphocyte division was stimulated with 10 μg/ml phytohemagglutinin (PHA). The lymphocyte culture was incubated for 50 h in the presence of PHA at 37°C. To accumulate metaphases, demecolcine at a concentration of 0.2 μg/ml was added in the culture 2 h before fixation. After the cultivation, the cells were treated with a hypotonic KCl solution (0.075 mol/l) for 15 min at 37°C, fixed with a mixture of methanol and glacial acetic acid (3 : 1), and placed onto glass slides. The dried smears were stained with azure eosin. The number of aberrant cells and the number of metaphases with chromosome aberrations were counted. For each donor, 100 metaphases were ana lyzed per one point. The spontaneous rate of the cells with single aberrations was zero to three cells per 100 analyzed cells. The micronucleus test was performed as recom mended by Albertini et al. [3] and Fenech et al. [4]. The fraction of the binucleate cells with MN was esti mated per 1000 analyzed binucleate cells, the cytoki nesis of which was stopped with cytochalasin B, i.e., the cells that passed through the first mitosis between 48 and 72 h after PHA stimulation. For this purpose, heparinized blood (0.7 ml; 15 U/ml) was supplemented with 3.3 ml of the RPMI medium containing 20% fetal bovine serum, 100 μg/ml streptomycin, 100 μg/ml penicillin, and 7 μg/ml PHA and incubated at 37°C. Cytochalasin B was added 48 h after the stimulation at a concentration of 6 μg/ml, and the cultivation was continued for addi tional 24 h. Then the cells were treated with hypotonic KCl solution (0.125 M), fixed with a mixture of meth anol and glacial acetic acid (3 : 1), and placed onto glass slides. The dried smears were stained with azure eosin. Two independent counters analyzed each prep aration. In the majority of cases, the damaged cell
contained one MN; sometimes cells with two or three MN were observed. Measurement of the Number of DNA DoubleStrand Breaks (DSBs) Lymphocytes were isolated from heparinized blood by centrifugation in Histopaque (Sigma) density gra dient according to the manufacturer’s protocol. After isolation, lymphocytes were washed and suspended in borate buffer (0.14 mol/l NaCl, 10 mmol/l Na2EDTA, 45 mmol/l H3BO3, and 14 mmol/l Na2B4O7; pH 8.3) to a final concentration of 1–2 × 106 cells/ml. The number of doublestrand breaks in the DNA of lymphocytes was determined by electrophoresis of single cells immobilized on agarose (DNA comet assay) [5, 6]. Briefly, 10 μl of cell suspension (12 × 106 cells/ml) were mixed with 100 μl of 0.5% low melting agarose (type IV) solution in phosphate buffer at a temperature of 37°C and loaded onto glass slides preliminary covered with 1% conventional agarose. The cells were lysed in cold (4°C) lysing buffer (2.5 mol/l NaCl, 100 mol/l EDTA, 20 mol/l TrisHCl pH 10.0, 1% Triton X100, and 10% DMSO) for 2 h. After cell lysis, the slides were transferred into cold (4°C) Trisborate buffer (TBE) pH 8.2 and kept for 20 min. Electrophoresis was conducted in TBE buffer at a voltage of 0.75 V/cm for 20 min at 4°C. After elec trophoresis, the slides were slightly dried and fixed with 70% ethanol for 10 min. DNA was stained with acridine orange (2 μg/ml in phosphate saline buffer pH 7.4. The DNA comets were visualized and recorded using the AxioVision 4.8 (Carl Zeiss) software in an Axioskop 40 FL (Carl Zeiss) luminescent microscope equipped with an MRс 5 (Carl Zeiss) digital camera. The microphoto images of DNA comets were analyzed and processed with the help of the СASP 1.2.2 (CASPlab) program. Estimation of ROS Production in Lymphocytes The content of reactive oxygen species in lympho cytes was assessed with the help of 5(and 6)chlorom ethyl2',7'dichlorodihydrofluorescein diacetate, DCF (Invitrogen). This preparation readily penetrates through the cell membrane, is hydrolyzed with esterases in the cell, and then is oxidized to fluorescent compound, presumably, by hydroxyl radical, peroxy radical, and peroxynitrite anion. The suspension of isolated lymphocytes in phosphate buffer pH 7.4 (200 μl; 10 × 106 cells/ml) was supplemented with 2 μl of 100 mmol/l DCF solution in DMSO and incubated at 37°C for 60 min to measure their fluorescence intensity in a Qubit (Invitrogen) portable fluorometer at an excitation wavelength of 488 nm and an emission wavelength of 525–530 nm. BIOPHYSICS
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Table 1. Comparison of the characteristics of the blood lymphocytes of cleanup workers with those of healthy individuals Initial frequency Examined of cells with aber cohorts rations, % Liquidators Population
1.381 ± 0.368 (21) 1.490 ± 0.181 (42) A
Number of aberrations per 100 cells chromosome 0.341 ± 0.129** (21) 0.022 ± 0.022 (42)
chromatid
Mean number Concentration of Initial rate of cells of DNA double reactive oxygen spe with micronuclei strand breaks, cies, arbitrary units (per 1000 cells) arbitrary units per 106 cells
1.463 ± 0.265 16.05 ± 1.64 (37) (21) 1.489 ± 0.180 13.33 ± 1.14 (12) (42)
14.42 ± 1.60** (21) 7.93 ± 1.10 (19)
0.405 ± 0.039** (21) 0.581 ± 0.048 (12)
Note: The number of examined individuals is in parentheses. ** p < 0.01 (Student’s ttest or Yatescorrected χ2test). A, the data on the rate of chromosome aberrations in unexposed population examined in 2006–2009 were used as a control.
Immunological Examination of the Immune Status (IS) This examination included determination of leu kocyte content, as well as the absolute (109 cells/ml) and relative contents of lymphocytes, including CD3+, CD4+, and CD8+ T lymphocytes; CD16+ lym phocytes; CD3+CD16/56+ (NKT) and CD3– CD16/56+ (NK); CD8+CD28+ lymphocyte; CD4+CD25+high regulatory T cells; and characteristics of cell activity (HLADR+, CD95+; activated T lym phocytes; CD19+ B lymphocytes; and the serum immunoglobulins of class M, G, and A and total IgE) [7]. The content of activated T lymphocytes was deter mined as the difference between the percentage rates of all HLADR+ lymphocytes and CD19+ B lympho cytes. The peripheral blood leukocytes and lymphocytes were counted in a Goryaev chamber using Zadorozh nyi dye. The phenotypes of subpopulations of lympho cytes and activation markers were determined by indi rect immunofluorescence with the help of monoclonal antibodies using a BD FACS II laser flow cytofluo rometer. The levels of serum IgM, IgG, and IgA were assessed according to a conventional radial immunod iffusion according to Mancini. Total IgE was deter mined using a HemaMedica kit for twosite (sand wich) enzyme immunoassay. Phagocytic activities of neutrophils and monocytes (PAN and PAM) in the blood serum without and with IgG opsonization were determined according to the protocol of N.S. Oliferuk using Staphylococcus aureus Wood 46 24h culture of the second inoculation (Tarasevich Institute of Stan dardization and Control) in a flow laser cytofluorom eter [8]. The measurement data were statistically processed, mean values were compared, and correlation analysis was performed using the Statistica software package. BIOPHYSICS
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RESULTS AND DISCUSSION Cytogenetic and Molecular Biological Characterization of Lymphocytes Metaphase analysis of the rate of the cells with chromosome aberrations has demonstrated that their rate in cleanup workers on the average does not differ from that in the control individuals (Table 1), amount ing to 1.381 ± 0.368 and 1.490 ± 0.181, respectively. The main type is chromatid aberrations; their rate in cleanup workers is 1.463 ± 0.265 (per 100 cells) and 1.489 ± 0.180 in the control population cohort. How ever, the rate of aberrations of chromosome type is sig nificantly higher in cleanup workers (0.341 ± 0.129 versus 0.022 ± 0.022). Note that one liquidator among the cohort of those involved in the emergency cleanup after the Cherno byl nuclear accident (the exposure dose of this liquida tor according to the dosimetric control was 22.3 rem) had a rate of chromatid aberration exceeding the num ber of such aberrations in the remaining examined individuals by one order of magnitude. This value was taken into account in the abovedescribed calculations but was discarded from correlation analysis. The rate of lymphocytes with micronuclei (MN) in cleanup workers varies in a very wide range, namely, from 2 to 52 cells with MN per 1000 examined cells and does not differ from the recorded exposure dose. The distribution of the examined cleanup workers according to the rate of the cells with MN is shown in Fig. 1. The mean value of the rate of cells with MN is 16.05 ± 1.64 and does not differ in a statistically signif icant manner from the control, 13.33 ± 1.14 (Table 1). For comparison, the mean value for the inhabitants of Moscow is 16.58 ± 1.09. However, it is evident that at least one value of the rate of cells with MN (between 50 and 55 or, more pre cisely, 52) is an exception. Verification has demon strated that this value falls beyond the 99.9% distribu tion interval. The exposure dose of this person was rather low, 0.5 rem. Presumably, such a high rate of the cells with MN is determined by his specific individual features (or an incorrectly measured dose of external γ irradiation). Therefore, this value (as “jumping out”) was discarded from the further analysis of the correla
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Comparison of the Immune Status of Cleanup Workers and Control Cohort
16 Number of observations
14
It has been demonstrated that the average lympho cyte contents in the cleanup workers and individuals of the control cohort in their relative (28.33 and 28.50%, respectively) and absolute (2.026 and 1.985 × 109 cells/l) values were almost the same. Then we compared the characteristics of the immune status using 28 studied parameters observed in the cleanup workers and the control population cohort.
12 10 8 6 4 2 0 –5 0 5 10 15 20 25 30 35 40 45 50 55 60 Number of cells with MN per 1000 examined cells Fig. 1. The distribution of the cleanup workers relative to the initial frequency of the lymphocytes with micronuclei (solid line shows the boundary of the corresponding theo retical distribution).
tions between the genome damage and parameters of the immune status. After removing this value, the mean rate of the cells with MN decreased to 15.05 ± 1.34 (Table 1). In general, the distribution fits a normal pattern (p = 0.30). Determination of the degree of DNA damage in the control cohorts and cleanup workers has demon strated that the level of DNA DSBs was higher in the cleanup workers in a statistically significant manner. It has also been found that the reactive oxygen species concentration in the lymphocytes of cleanup workers is significantly lower as compared with the lympho cytes of control population cohort (Table 1).
Table 2 lists the values of the parameters differing in these two groups. In particular, the mean percentage content of cytotoxic CD8+ lymphocytes, CD16+ nat ural killers, and CD3+CD16+CD56+ NKT cells (dis playing antivirus and antitumor activities and destroy ing the virusinfected and tumor cells) increased in cleanup workers in a statistically significant manner as compared with the control. The mean percent rate of HLADR+, a late lymphocyte activation marker, and the content of regulatory T lymphocytes (CD4+CD25+high) also elevated. On the other hand, the control cohort displayed a significantly higher index of immune regulation (CD4+/CD8+) as com pared with the cleanup workers due to a decrease in the content of cytotoxic CD8+ lymphocytes. Both groups had very high values of phagocytic activities of neutrophils and macrophages (PAN and PAM), yet these values in cleanup workers were significantly lower. Several characteristics of humoral immunity also changed; in particular, the level of serum IgA was significantly elevated in cleanup workers.
Table 2. Mean values of the immune status characteristics of cleanup workers that differ from the corresponding charac teristics of unexposed population Characteristics of immune status
Liquidators M ± me
Control group M ± me
p
CD4+ T lymphocytes, % CD8+ T lymphocytes, % CD4/CD8 CD16+ lymphocytes, % CD3+CD16+CD56+, % CD3 CD16+CD56+, % CD4+CD25+high, % HLADR+, % Activated T lymphocytes, % PAN, % PAM, % IgA, g/l
41.690 ± 1.75 31.440 ± 1.80 1.559 ± 0.130 19.11 ± 2.23 15.730 ± 1.55 17.983 ± 1.39 3.321 ±s 0.139 16.490 ± 1.03 7.310 ± 0.715 62.153 ± 4.91 54.69 ± 3.97 2.70 ± 0.16
47.271 ± 2.37 23.769 ± 2.21 2.182 ± 0.211 12.200 ± 1.36 9.586 ± 0.938 11.979 ± 1.11 2.743 ± 0.193 11.871 ± 0.760 4.936 ± 0.405 81.96 ± 3.85 68.89 ± 4.22 1.67 ± 0.24
0.092 0.029 0.019 0.051 0.027 0.018 0.031 0.013 0.060 0.020 0.045 0.001
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5.5
r = 0.490, p = 0.024
Content (%) of activated T lymphocytes
Content (%) of CD4+CD25+high lymphocytes
6.0
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5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 0
5
10 15 20 25 30 Number of lymphocytes with MN per 1000 examined cells
35
12 10 8 6 4 2 0.1
0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Reactive oxygen species concentration in lymphocytes, arbitrary units
Fig. 2. The correlation between the frequency of the peripheral blood lymphocytes with micronuclei and the content of CD4+CD25+high lymphocytes.
Fig. 3. The correlation between the reactive oxygen species concentration in lymphocytes and the content of activated T lymphocytes.
The Correlation between Genome Damage and Immune Status
T lymphocytes: 7.310 ± 0.715 versus 4.936 ± 0.405 in the control population (p = 0.061). The cause of this discrepancy is still unclear. A larger number of correlations is detectable as a sufficiently reliable trend at p < 0.10 (Table 3). For example, note the correlations between PAN and the parameters characterizing the cell genome damage (Table 3), namely, that the mean relative PAN value displayed by cleanup workers is lower than the control and decreases proportionally to an increase in the rate of the cells with MN or the level of DNA DSBs (p = – 0.313, p = 0.067 and r = –0.375, p = 0.094, respec tively). Despite the small number of chromosome aberra tions in the lymphocytes of cleanup workers, it is likely that their appearance led to an increase in the number of CD3–D16+CD56+ (NK), since an almost statisti cally significant correlation between these parameters is observed (r = 0.421, p = 0.072). The increase in DNA DSBs in cleanup workers as compared with the common population (Table 1) is a cause underlying the observed increase in the number of CD3+CD16+CD56+ lymphocytes: the correlation coefficient between these parameters is 0.384 (p = 0.085). As is evident from Table 2, the immune status of cleanup workers differs from that of the unexposed population in the mean values of 12 examined param eters. Thus, we have demonstrated that the genomic instability induced 24 years ago in the stem cells and precursors of lymphocytes by exposure to ionizing radiation during the emergency cleanup after the Chernobyl nuclear accident is currently detected as damage to the cell genome and changes in several
No statistically significant correlations of the recorded radiation dose received by cleanup workers, DNA DSB level, and the frequency of chromosome or chromatid aberrations with the characteristics of the immune status were observed. The detected correla tions of the genome damage in the lymphocytes of cleanup workers and characteristics of their immune status are described below (Fig. 3). A mediumstrength correlation between the con tent of regulatory T lymphocytes (CD4+CD25+high) and the rate of cells with MN (r = 0.429, p = 0.010). If the data for two individuals with the rate value of 34 (falling to the boundary of a 95% interval) are dis carded from the analysis, then the correlation coeffi cient increases to 0.620 at p = 0.0001 (Fig. 2). The trend of the correlation between this characteristic and the rate of chromatid aberrations is also observed but in the opposite direction: it decreases with an increase in the rate of chromatid aberrations (r = –0.436, p = 0.070). Thus, the first dependence is likely to domi nate. A statistically significant correlation between the reactive oxygen species concentration in the lympho cytes of cleanup workers and the content of activated T lymphocytes has been detected: the content of these lymphocytes increases with the reactive oxygen spe cies concentration (r = 0.49, p = 0.024; Fig. 3). How ever, since the ROS concentration in the lymphocytes of cleanup workers is significantly lower than that in the control (Table 1), it could be expectable that the content of activated T lymphocyte in the blood is also decreased. However, the cleanup workers display a trend of an increase in the percent of activated BIOPHYSICS
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Table 3. The detected correlations Correlations Statistically significant Frequency of cells with MN–CD4+CD25+high Reactive oxygen species concentration–activated T lymphocytes Trends + Frequency of chromosome aberrations–CD16 Rate of chromosome aberrations–CD3–CD16+CD56+ Rate of chromatid aberrations–CD4+CD25+high Rate of chromatid aberrations–CD4+abs Frequency of cells with MN–PAN Level of DNA DSBs–CD3+CD16+CD56+ Level of DNA DSBs–PAN
immunological characteristics of the cleanup workers. This is especially evident from the example of regula tory T lymphocytes (CD4+CD25+high), whose relative content considerably increases with the degree of damage in the lymphocyte genome (rates of the lym phocytes with MN). Interestingly, an analogous dependence has been earlier described for radiologists [9]. An increase in the content of regulatory T lym phocytes enhances inhibition of the antitumor immu nity, thereby increasing the risk of cancer development in the distant future. In general, the results of this study suggest antivirus and antitumor immunity stress in the cleanup workers as compared with the unex posed population control; the cleanup workers display an activated type of immune system as compared with the control cohort. The detected trend for the growth in the content of CD16+ lymphocytes in the individuals that partici pated in the emergency cleanup after the Chernobyl nuclear accident with the increase in the frequency of lymphocytes with chromosome aberrations is a char acteristic of early aging. Note also that the decreased oxidative status of the lymphocytes of cleanup workers can lead to hypoxia, ischemia, infarctions, strokes, and other diseases.
r
p
0.620 0.490
0.0001 0.024
0.421 0.421 –0.436 –0.468 –0.313 0.384 –0.375
0.072 0.072 0.070 0.050 0.067 0.085 0.094
ACKNOWLEDGMENTS The work was supported by the Russian Founda tion for Basic Research (grant no. 090400347a). REFERENCES 1. V. A. Shevchenko and G. P. Snigireva, Radiats. Biologiya Radioekologiya 46 (2), 133–139 (2006). 2. D. A. Hangesford, Stain Techn. 40, 333–338 (1965). 3. R. J. Albertini, G. R. Douglas, et al., Mutat. Res. 463, 111–172 (2000). 4. M. Fenech, W. P. Chang, M. KirschVolders, et al., Mutat. Res. 534, 65–75 (2003). 5. N. P. Singh, M. T. McCoy, R. R. Tice, and E. L. Schnei der, Exp. Cell Res. 175, 184–191 (1988). 6. I. I. Pelevina, M. M. Antoshchina, V. A. Bondarenko, et al., Radiats. Biologiya Radioekologiya 47 (2), 141– 150 (2007). 7. R. V. Petrov, R. M. Khaitov, B. V. Pinegin, and I. V. Ora dovskaya, Immunologiya, No. 6, 51–62 (1992). 8. N. S. Oliferuk, Optimization of the Methods for Estima tion of the Phagocytic Activity of Peripheral Blood Leuko cytes by Laser Flow Cytometry, Extended Abstract of Candidate’s Dissertation in Medical Sciences (Mos cow, 2005). 9. E. Torkabadi, A. Kariminia, and F. Zakeri, Iran J. Allergy Asthma Immunol. 6 (4), 181–187 (2007).
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