Relationships between a Thiol-Disulfide System and Liposoluble Antioxidants with Cytogenetic Indices in Humans Exposed to Low Doses Radiation

doi:10.4236/eng.2013.510B013

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Engineering, 2013, 5, 62-67

http://dx.doi.org/10.4236/eng.2013.510B013 Published Online October 2013 (http://www.scirp.org/journal/eng)

Relationships between a Thiol-Disulfide System and

Liposoluble Antioxidants with Cytogenetic Indices in

Humans Exposed to Low Doses Radiation

G. F. Ivanenko, E. B. Burlakova

Russian Academy of Sciences, Emanuel Institute of Biochemical Physics, Moscow, Russia

Email: [email protected], [email protected]

Received October 2012

ABSTRACT

This work presents the results of biochemical examination of people exposed to irradiation as a result of the Chernobyl

catastrophe. In remote period ( in 4, 5, 6 and 7 years ) after Chernobyl accident we stud ied the state of the redox syste m

of glutathione(GSH, GSSG) and the response of the system of essential lipid antioxidants (vitamin E, A) in b lood plas-

ma of people of various ages. An analysis of correlations between cytogenetic indices in lymphocytes and levels of re-

duced glutathione and liposoluble antioxidants in the plasma of peripheral blood in children born after the Chernobyl

accident and liquidators is presented. The cumulative doses for the examined group of children received by their moth-

ers from 0.8 to 70 cSv and liquidators received, on aver age, the highest irradiation doses from 0.1 to 150 cSv. A com-

plex relationship between lipo-and water-soluble antioxidants level in plasma in human population (children living in

radionuclide-contaminated regions and the Chernobyl liquidators) exposed to chronic low-level radiation after the

Chernobyl accident was demonstrated. The obtained experimental data indicate different responses of the human popu -

lation water-and fat-soluble antioxidants system to low (from 0.1 to 20 cSv) and high (from 20 to 150 cSv) doses of

ionizing radiation.

Keywords: Antioxidant; Radiation; Pla sma ; Human Population

1. Introduction

A large number of new facts concerning the effect of

low-level radiation on living organisms were provided

after the Chernobyl accident. The significance of low-

dose exposures for cell survival, tissue damage, and indi-

vidual health are poorly understood [1,2]. For a long pe-

riod of nuclear industry development, exposures to low

doses were considered safe. However, recent publica-

tions demonstrate a relatively high efficiency of low-

level radiation in the region of low radiation doses. Ac-

tive mutation process in various cells and tissues and

carcinogenesis promotion were observed after exposure

to low radiation doses [3]. Mutagenesis is accompanied

by chromosomal and genomic instability in cells of vari-

ous tissues. In the case of ionizing radiation, the primary

damage, namely double-strand breaks, induced both

structural chromosomal aberrations and microstructural

DNA damage detected as gene mutations [4].

Various biochemical and biophysical tests are being

developed in order to evaluate the effect of exposure to

low radiation doses; in particular, the antioxidant status

of the body is given considerable attention. Glutathione

is particularly important among water-soluble antioxi-

dants [5,6]. Resistance of the body to ionizing radiation

depends on both lipid antioxidant activity and the level of

sulfhydryl groups [7,8].

It is common knowledge th at thiols as well as vitamins

C, A, and E p rotect cells from oxidation and inhibit lipid

peroxidation. A relatively high contribution of water-

soluble antioxidants is commonly associated with rege-

neration. Glutathione was proposed to mediate the reduc-

tion of at least two vitamins, α-tocopherol and ascorbic

acid; it increases their effect and prevents lipid peroxida-

tion [9,10].

Below we present experimental data on the effect of

low-level radiation on the status of water-and lipo-so-

luble antioxidant in the liquidators and people living in

conditions of long-term exposure to low radiation doses

in the regions contaminated by radionuclides after the

Chernobyl accident (1 - 20 Ci/km2). We tried to establish

the relationship between levels of glutathione, v itamin E,

A and the level of radiation burden and to analyze the

dose-response relationships for certain types of genetic

damages such as the frequency chromatid breaks, in the

course of long-term monitoring. Below we analyze poss-

ible correlations between cytogenetic indices in peri-

G. F. IVANENKO, E. B. BUR L AKOVA

pheral blood lymphocytes, glutathione and lipid antioxi-

dant levels in blood plasma of people after the Chernobyl

accident.

2. Methods and Subjects

Children were examined from birth to the age of eight in

three regions with different levels of soil pollution with

radionuclides (largely 137Cs) after the Chernobyl accident.

Children (n = 125) from the Chechersk Region (15 - 20

Ci/km2) (Gomel Region of Belorussia) were examined 5

years after the accident; after six years children (n = 210)

from the Mtsensk Region (1 - 5 Ci/km2) and, after seven

years, children (n = 116) from the Bolkhov Region (5 -

10 Ci/km2) (both Orlov Region of Russia) were ex-

amined. Children at the age of 5 - 6 - 7 years (at the mo-

ment of examination) survived the accident during their

intrauterine development; other ones were born after the

accident (1990-1992).

Four years after the Chernobyl accident (1986), we

examined the liquidators (LI) that were working in the

first days after the accident and later moved to Slavutich

town outside the 30 km zone (n = 22). The radiation

doses of this group ranged from 2 to 150 cSv. Six years

later, we examined one more group of liquidators (L2, n

= 128) who worked fro m May 1986 to 1987 in the region

of Chernobyl accident and received radiation doses from

0.1 to 70 cSv. Were examined сhildren at the age 2 - 12

(n = 9) and of adults of Slavutich (n = 29) living in ra-

dionuclide-contaminated regions (1 - 5 Ci/km2). Resi-

dents of Moscow (n = 21) not exposed to radiation were

the comparison group.

The level of reduced and oxidized glutathione in plas-

ma was determined as described elsewhere [11,12] with

modifications. Glutathione level was determined in 20 -

100 µl plasma by spectrofluorometry at 350/420 nm. The

level of oxidized glutathione was calculated as the dif-

ference between the total numbers of SH groups before

and after the reduction using blank assays and standard

curves fo r GSH and G SSG . All results averaged for three

parallel experiments were expressed as µM. Tocopherol

and retinol were determined by spectrofluorometry at

295/325 and 330/470 nm, respectively, with appropriate

standards as desc ribed [13] .

The cytogenetic test relied on the traditional method

(analysis of unstable chromosomal aberrations). Whole

blood (0.5 ml) was cultivated as described by [14]. Both

chromosomal (dicentrics, centric rings, and paired frag-

ments) and chromatid (single fragments) aberrations

were evaluated. The fr equency of chr omatid b reaks (FC B)

calculated as a sum of single fragments (Fs), doubled

paired fragments (Fp), quadrupled dicentrics (Dc), and

rings (Cr) per 100 m e taphases .

Individual radiation doses of the Chernobyl liquidators

were determined as a total Chernobyl radiation loading.

Individual accumulated doses of radiation were deter-

mined from the age of children or as a total Chernobyl

radiation burden of their mothers before delivery. We

studied the effect of accumulated radiation dose of

mothers (Dm) after the Chernobyl accident on individual

cytogenetic and biochemical changes in peripheral blood

of their children.

3. Results and Discussion

Table presents the mean level of water-and lipo-soluble

antioxidants (GSH, vitamins E and A), oxidized gluta-

thione in blood plasma of adults and children living in

uncontaminated and radionuclide-contaminated regions

after the Chernobyl accident (1 - 20 Ci/km2 by l37Cs) as

well as of the liquidators with th e radiation doses from 2

to 150 and from 0.1 to 70 cSv (group s LI and L2, respec-

tively). Table demonstrates the absence of significant

differences in the studied indices in children and adults

living in regions with different radionuclide contamina-

tion. Only the liquidators (LI) had a significantly lower

GSH and a significantly higher level of GSSG in the

blood plasma as compared to all studied residents (Table

1). However, the stability of the blood plasma indices in

children from Mtsensk (1 - 5 Ci/km2), Bolkhov (5 - 10

Ci/km2), and Chechersk (15 - 20 Ci/km2) with different

levels of soil contamination by radionuclides was consi-

derably lower as compared to people from uncontami-

nated regions. The χ2 test demonstrated that GSH level

follows the Poisson distribution. Poisson variables have

particularly high coefficients of variatio n (CV).

Table 1 shows that the CV exceeds 40% for GSH in

children living in these regions. The highest individual

variability was observed in children from the Chechersk

region with the highest radionuclide contamination (15 -

20 Ci/km2); the corresponding CV reflecting variability

of the indices was equal to 82% for GSH and 67% for

vitamin A. Levels of tocopherol and retinol in children

from Chechersk region was significantly lower than in

residents of uncontaminated regions. In addition to lipo-

peroxidative stress, children of Chechersk had a three

time s higher levels of GSSG in the blood plasma in

comparison to Mtsensk and of Bolkhov residents (Table

1).

Apparently, the high individual variability of the stu-

died indices in children depends on radiobiological prop-

erties of ionizing radiation in case of long-term exposure

of their mothers to low-level radiation. We studied the

effect of accumulated total radiation doses of mothers

(Dm) after the Chernobyl accident on long-term changes

of biochemical and cytogenetic indices in their children

in comparison with the dose effects in the Chernobyl

liquidators.

G. F. IVANENKO, E. B. BUR L AKOVA

Table 1. Mean plasma levels of reduced, oxidized glutathione, tocopherol and retinol in children and adults living in un-

contaminated and radionuclide-contaminated regions [Mean ± SEM (SD)].

Children

Mtsensk, 1 - 5 Ci/km2 Bolkhov, 5 - 10 Ci/km2 Chechersk, 15 - 20 Ci/km2 Slavutich, 1 - 5 Ci/km2

Age, years 1.7 ± 0.1 3.0 ± 0.2 0.8 ± 0.1 6.3 ± 0.8

GSH, µM 20.5 ± 0.7 (10.3) 18.6 ± 0.9 (9.6) 21.1 ± 1.6 (17.4) 16.0 ± 2.3 (6.9)

n 218 116 125 9

Min - Max 4.8 - 61.6 5.0 - 57.4 1.6 - 101.7 4.9 - 28.1

CV,% 50 52 82 43

GSSG, µM 11.3 ± 0.4 (5.9) 9.9 ± 0.4 (4.1) 32.1 ± 1.3 (14.7) 44.2 ± 6.6 (19.7)

n 210 116 120 9

Min - Max 0.8 - 38.6 1.4 - 22.7 3.3 - 69.4 17.0 - 70.3

CV,% 52 41 46 45

Vitamin E, µM 9.0 ± 0.3 (4.5) 21.4 ± 0.9 (7.8) 8.3 ± 0.4 (4.2) 8.4 ± 0.8 (2.4)

n 198 67 120 9

Min - Max 0.7 - 45.6 7.3 - 37.8 0.7 - 27.9 4.9 - 12.6

CV, % 50 36 51 29

Vitamin A, µM 0.9 ± 0.03 (0.5) 2.4 ± 0.1 (1.2) 0.9 ± 0.1 (0.6) 2.0 ± 0.2 (0.5)

n 197 67 117 9

Min - Max 0.2 - 5.7 0.6 - 5.8 0.1 - 3.4 1.5 - 3.9

CV, % 55 50 67 25

Adults

Liquidat or s 1, 2 - 150 cSv Liquida tor s 2, 0.1 - 70 cSv Slavutich 1 - 5 Ci/km2 Moscow

Age, years 42.0 ± 1.8 45.6 ± 1.0 40.7 ± 2.4 39.2 ± 2.7

GSH, µM 15.8 ± 1.2 (5.8) 24.8 ± 0.9 (9.7) 33.4 ± 2.9 (15.4) 19.4 ± 1.6 (7.4)

n 22 128 29 21

Min - Max 6.9 - 26.5 6.7 - 55.2 13 - 68.6 11.2 - 40.0

CV,% 37 39 46 38

GSSG, µM 30.9 ± 1.5 (7.3) 16.8 ± 1.0 (8.8) 40.4 ± 1.8 (9.7) 13.4 ± 1.5 (6.3)

n 22 81 29 19

Min - Max 16.7 - 44.6 3.6 - 54.2 19.3 - 61.9 5.5 - 27.1

CV,% 24 52 24 47

Vitamin E, µM 22.7 ± 1.2 (5.7) 19.4 ± 0.7 (7.2) 13.6 ± 1.1 (5.6) 22.3 ± 1.6 (7.4)

n 22 120 29 21

Min - Max 15.0 - 39.0 5.1 - 39.5 7.2 - 30.1 8.1 - 40.5

CV, % 25 37 41 33

Vitamin A, µM 3.5 ± 0.3 (1.2) 2.9 ± 0.1 (1.2) 2.6 ± 0.2 (1.2) 2.9 ± 0.2 (0.7)

n 22 120 29 21

Min - Max 1.8 - 5.9 0.5 - 7.9 1.1 - 6.9 2.0 - 4.4

CV, % 34 41 46 24

Figures 1(a)-(e) present the dose dependence levels

water-and lipo-soluble antioxidants (GSH, tocopherol,

retinol), oxidized glutathione in the plasma as well as the

frequencies of chromatid breaks in peripheral blood

lymphocytes of children living in Mtsensk (with radio-

nuclide contamination of the soil within 1 - 5 Ci/km2; n =

35), Bolkhov (5 - 10 Ci/km2; n = 26 ) , and Chechersk (15

- 20 Ci/km2; n = 31) as well as of the LI liquidators that

worked in the first days after the accident (n = 22) and

the L2 liquidators (n = 128) that worked from May 1986

to 1987 in the region of the Chernobyl accident with total

radiation doses from 0.1 to 150 cSv.

G. F. IVANENKO, E. B. BUR L AKOVA

Dose, cSv

-20 020406080100 120 140 160

GSSG, µM

50 b

Dose, cSv

-20 020406080100 120 140 160

Retinol, µM

Dose, cSv

-10 010 20 30 40 50 60 70 80

Frequency of chromatid breaks, %

-2

Figure 1. Relationship between total radiation exposure, plasma levels of reduced (a) and oxidized (b) glutathione, tocopherol

(c), retinol (d), frequencies of chromatid breaks (e) in peripheral blood lymphocytes in children living in regions with different

radionuclide contamination as well as in the Chernobyl liquidators (individual indices): 1-Mtsensk, 1 - 5 Ci/km2; 2-Bolkhov, 5 -

10 Ci/km2; 3-Chechersk, 15 - 20 Ci/km2; 4-liquidators 1, 2 - 150 cSv; 5-liquidators 2, 0.1 - 70 cSv; 6-“control”-comparison

group. In Figure 1(e), there is marked a range of examined individuals with levels of frequencies of chromatid breaks which

correspond with t h e l ev el o f “ control”.

Note that radiation burden of mothers living in the

most radionuclide-contaminated Chechersk region (0.8 -

60 cSv) were similar to those of the L2 liquidators (0.1 -

70 cSv) who had worked in the region of the Chernobyl

accident. The pattern of the relationship was similar. The

level of reduced glutathione in the plasma increased in

certain individuals exposed to doses below 20 cSv and

decreased for the doses from 20 to 60 cSv (Figure 1(a))

Dose, cSv

-20 020406080100 120 140 160

GSH, µM

100

120

1-Mtsensk, 1-5 Ci/km

2-Bolkhov, 5-10 Ci/km

3-Chechersk, 15-20Ci/km

4-liquidators 2, 0,1 - 70 cSv

5-liquidators 1, 2 -150 cSv

6-"control"-comparison group

0.1

Dose, cSv

-20 020406080100 120 140 160

Tocopherol, µM

G. F. IVANENKO, E. B. BUR L AKOVA

Further increase in the radiation burden to 20 - 150 cSv

increased GSSG level in the plasma (Figure l(b)) and

decreased GSH level in the plasma (Figure l(a)), which

decreased the glutathione potential. Decreased levels of

reduced glutathione under the influence of low-level

radiation (below 20 cSv) is accompanied by accelerated

consumption of vitamin E (Figure 1(c)) and increased

level of vitamin A in blood plasma (Figure 1(d)). The

deficiency of lipo-and water-soluble antioxidants reveal-

ed long after the accident (4 - 7 years) in the liquidators

and children of exposed mothers is an important health

marker for populations exposed to chronic low-level in

radiation. The system of water- and liposoluble antioxi-

dants differently responses to radiation exposure of low

intensi t y depending on the ex posure dose of ra diatio n.

Different responses to the studied systems in human

population to the effect of low-level radiation can be due

to an increased sensitivity of the systems to radiation

resulting from disturbed control mechanisms involving

free radicals. High stationery concentrations of active

oxygen forms at the low doses confirm proposed “delay”

of the repairer systems [15]. Low efficiency of the re-

pairer systems increase the level of spontaneous chro-

mosomal disturbances, which induce mutations in genes

and increase the incidence of cancer [16].

This conclusion is confirmed by an increase in cyto-

genetic disturbances in lymphocytes of peripheral blood

determined upon an individual examination of this group

of people (children and liquidators). Figure 1(e) presents

the individual dose dependence of the frequencies of

chromatid breaks in peripheral blood lymphocytes of

children (Mtsensk, 1 - 5 Ci/km 2; Bolkhov, 5 - 10 Ci/km2;

and Chechersk, 15 - 20 Ci/km2) living in regions with

different levels of radionuclide contamination of the soil

after the Chernobyl accident as well as of liquidators 2,

(0.1 to 70 cSv.) These experiments demonstrate the com-

plex dependence of the frequencies of chromatid breaks

in lymphocytes in human population on radiation doses.

For instance, at low radiation doses (from 0.8 to 30 cSv),

some individuals have a considerably increased frequen-

cies of chromatid breaks (up to 12 per 100 cells; F igure

1(e)) in lymphocytes as compared to those in people not

exposed to ionizing radiation. A two-or three-fold in-

crease in the cytogenetic indices is observed in over 20%

people at low radiation doses. When the doses increased

from 30 to 60 c Sv, the frequencies of chromatid breaks in

peripheral blood lymphocytes also increased (up to 5 per

100 cells; Figure 1(e)). A 3 - 5-fold deviation of indi-

vidual cytogenetic and biochemical indices from the

control (Figure 1) indicates a high rate of mutations in

somatic cells of children and L2 even after a long period

after the accident (5 - 7 years). This fact can be consi-

dered as an epidemiological confirmation of the role of

chromosomal aberrations in carcinogenesis, since the

Figure 2. Relationship between frequencies of chromatid

breaks in peripheral blood lymphocytes and plasma levels of

reduced glutathione, in children living in regions with dif-

ferent radionuclide contamination as well as in the Cher-

nobyl liquidators 2 (individual indices). In Figure 2, there is

marked a range of examined individuals with levels of fre-

quencies of chromatid breaks and levels GSH of people

which correspon d with the level of “ co n t ro l” .

risk of cancer increases in individuals with high level of

spontaneous chromosomal aberrations [16].

It was substantiated the studies of antioxidant status

and role of cytogenetic examinations for predicting of the

delayed consequences of irradiation. The development of

various biochemical and the cytogenetic tests are of great

importance for evaluation and prediction of individual

radiosensitivity of the organism.

As an example, let us present data on the correlation

between cytogenetic indices in peripheral blood lym-

phocytes and plasma levels GSH of people exposed to

low doses of radiation (Figure 2). We observed unidirec-

tional and bidirectional changes frequencies of chromatid

breaks and biochemical indices of human blood (positive

and negative correlation).

Quantitatively different response of the body to low

(below 20 cSv) and high (from 20 to 150 cSv) doses of

ionizing radiation is an unbiased marker of radiation-

induced disorders and underlies development of far dis-

orders in human population. To the great probability, the

low-rate radiation possesses another mechanism com-

pared wit h hi gh doses of a c ute radia tion.

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Frequency of chromatid breaks, %

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