Cytogenetic damage in human blood lymphocytes exposed in vitro to radon

Mutation Research 661 (2009) 1–9

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Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres

Cytogenetic damage in human blood lymphocytes exposed in vitro to radon V. Zareena Hamza, Mary N. Mohankumar ∗ Radiological Safety Division, Indira Gandhi Centre for Atomic Research (IGCAR), Kalpakkam-603 102, Tamilnadu, India

a r t i c l e

i n f o

Article history: Received 2 July 2008 Received in revised form 14 October 2008 Accepted 17 October 2008 Available online 30 October 2008 Keywords: Radon In vitro irradiation Chromosome aberration Micronuclei Nuclear bud Nucleoplasmic bridges High LET radiation

a b s t r a c t The effect of radon in inducing DNA damage was investigated in vitro by two well-established cytogenetic assays. Blood samples were irradiated with radon using a novel irradiation assembly. Doses varied between 0 and 127 mGy for chromosome aberration (CA) assay and 0 and 120 mGy for cytokinesis blocked micronucleus (CBMN) assay. Dose-rates varied between 0.000054 and 0.708 mGy/min. After the irradiation period of 3 h, excess radon gas was released and cultures were initiated using standard procedures. Chromosome aberrations such as dicentrics, excess acentric fragments, acentric rings, centric rings, chromatid breaks were observed. Micronuclei, nucleoplasmic bridges and nuclear buds were scored by the CBMN assay. A significant increase in the frequency of dicentrics, excess acentric fragments and centric rings was observed with increasing radon dose, whereas total acentric rings plus double minute and chromatid breaks/cell were not significantly elevated. In CBMN assay, the frequency of micronuclei was found to be significantly raised whereas that of nucleoplasmic bridges and nuclear buds were not. Nucleoplasmic bridges and nuclear buds tended to increase with dose but did not achieve statistical significance. There was a strong positive correlation between nucleoplasmic bridges and dicentrics (P < 0.028) or rings (P < 0.0001) and between micronuclei and acentric fragments (P < 0.0005). The study shows that radon is capable of inducing significant chromosome damage at very low doses and dose-rates. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Radon is an inert, radioactive gas that decays to produce alpha particle emitting progenies of great concern to the environment. Alpha particles are high linear energy transfer (LET) radiations and carry sufficient energy to cause permanent damages to DNA and when compared to gamma radiation, do not penetrate deeply into tissues [1]. Epidemiological evidences such as cohort studies of underground miners with relatively high level of radon exposure, case control studies, etc., have shown that exposure to high levels of radon leads to lung cancer. The biological effects of radon are predominantly due to the alpha particle activity of 222 Rn and two of its solid decay products 218 Po and 214 Po. Inhalation of radon results

Abbreviations: CA assay, chromosome aberration assay; CBMN assay, cytokinesis block micronucleus assay; LET, linear energy transfer; BN, binucleated; MN, micronucleus or MNi micronuclei; NPB, nucleoplasmic bridges; NBUDs, nuclear buds. ∗ Corresponding author. Tel.: +91 44 27480500x23439; fax: +91 44 27480235. E-mail addresses: [email protected], [email protected] (M.N. Mohankumar). 0027-5107/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2008.10.010

in the deposition of 218 Po and 214 Po in the lungs damaging the cell lining of the airways and cause lung cancer [2]. Personal exposure to radon varies depending on the concentrations present at homes or in the occupational environment. Based on evidences from human and animal exposures, the International Agency for Research in Cancer (IARC) has classified radon as a group 1 carcinogen [3]. Animal experiments have clearly shown an increase in respiratory tract tumors due to inhalation of radon and its progenies [4–7]. However, variation in tumor incidence suggest that speciesto-species extrapolations of absolute risk is unlikely to be useful. In addition, a number of in vitro studies using radon were defined for understanding the early changes induced at cellular and molecular level and DNA repair of these events. Such studies are important in understanding the response of cells to environmental radon where only a small fraction of the cell population interacts with alpha particles [8]. Since equal doses of different types of radiations produce different biological effects, the differences in biological response per unit “dose” are accommodated by the application of a radiationweighting factor termed Relative Biological Effectiveness (RBE). Although a radiation-weighting factor of 20 has been designated

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for alpha particles, uncertainty exists on a realistic value of the RBE of alpha radiation. The risk per unit dose for alpha radiation is estimated to be anywhere between 2.5 and 20 times higher than that for high acute doses of low-LET radiation. Moreover, the RBE of alpha-emitters tends to increase as the dose decreases, probably due to the decreased effectiveness per Gy of low-LET radiation at low doses and low-dose rates [9]. Besides, from the radiation protection point of view, there is a serious concern that the RBE for alpha-emitters might be substantially underestimated [10], as it neglects the small, but potentially significant, ionization caused by the recoil of the parent nucleus during the alpha decay. However, for lung cancer induction after inhalation of short-lived radon progenies, a radiation-weighting factor of 10 was found to be more realistic than the value 20 proposed earlier [11]. To tackle uncertainties in determining the RBE of alpha particles, there is a need to conduct experiments in vitro wherein confounding factors such as dose, dose-rate, etc., could be controlled and monitored. A major difficulty in pursuing experiments with high LET alpha particles is due to their very limited ranges [12,13]. Causes of uncertainty include the fact that RBE of alpha is significantly dependent on the tissue, the endpoint, dose and dose-rate. Further, studies have shown that exposure to alpha particles can result in “bystander effects”, defined as the induction of biological effects in cells that are not directly traversed by a charged particle, but are in close proximity to them. As a result of the bystander effect, a linear back extrapolation of risks at high doses to low doses may significantly underestimate the risks at low doses [14]. Identifying an appropriate biomarker and relating it to energy of ionizing radiation would be an alternate approach to determining a realistic RBE in radiation protection. Sensitive biomarkers of DNA damage are required to resolve ambiguities related to RBE and bystander effects. In vitro studies have the advantage that it is not be confounded by demographic, life-style factors, etc. Cytogenetic biomarkers have proved to be excellent methods to detect and estimate radiation induced DNA damage. These include chromosomal aberrations, gene mutations, micronuclei assays, etc., among these, chromosome aberrations are regarded the most sensitive biomarkers exhibiting a dose response and have widely used in biological dosimetry. The cytokinesis blocked micronucleus (CBMN) is a versatile and simple method to estimate DNA damage and cytotoxicity. DNA damage events are scored specifically in once-divided binucleated (BN) cells and include (a) micronuclei (MNi), a biomarker of chromosome breakage and/or whole chromosome loss, (b) nucleoplasmic bridges (NPBs), a biomarker of DNA misrepair and/or telomere end-fusions, and (c) nuclear buds (NBUDS). The CBMN assay has also been successfully applied for dose estimation in cases of accidental overexposure to ionizing radiations [15,16]. Dose–response relationships for both high- and low-LET radiations have been well established using this assay [17]. MNi have proved to be sensitive biomarkers of radon exposures in miners and occupationally exposed personnel [18,19]. Micronuclei induction was observed in rat deep-lung fibroblasts exposed to radon in vitro as well as in vivo [20]. Of late, the CBMN assay incorporates scoring of nucleoplasmic bridges and nuclear buds in addition to micronuclei [21]. Scoring of nucleoplasmic bridges is aimed at understanding chromosomal rearrangements, which otherwise is not measured in the CBMN assay; as for example, most of the NPBs are thought to be formed when the centromeres of dicentric chromosomes are pulled to opposite poles at anaphase. In an earlier study we have successfully demonstrated the use of a custom-designed, simple and novel assembly to irradiate blood samples with radon and its progeny. The method is cost-effective,

safe and facilitates study of both environmental and high background level concentrations of radon. The efficacy of the method in producing chromosome aberrations in blood lymphocytes was established [22]. In the present study, samples were exposed to radon doses up to a maximum of 127 mGy and DNA damage was assessed by the Giemsa-stained chromosome assay and the CBMN assay. Nucleoplasmic bridges and nuclear buds were scored in addition to micronuclei in radon exposed samples and the results compared and co-related. 2. Materials and methods 2.1. Sample collection Blood samples were collected using heparinised vacuette tubes (Greiner labortechnik, Austria) from healthy non-smoking individuals working at of the Indira Gandhi Centre for Atomic Research, after obtaining informed consent. About 10 ml of blood sample each aliquoted were transferred to two 100 ml glass bottle. One served as control and the other exposed to radon. 2.2. Radon source A Radon source with an activity of 98.9 kBq was procured from Pylon (Model RN-1025, Canada). The source constitutes of dry Ra-226 in powdered form enclosed between a glass and plastic filter in the source container, and calibrated by Pylon against National Institute of Standards and Technology (NIST) certified radium liquid standards [23]. A pre-filter of 0.8 ␮m pore-size ensures that solid radon progeny do not enter into the irradiation chamber. 2.3. Irradiation A three-way cock, used for passing blood, saline and glucose, was used for evacuation of air from the bottle containing blood sample and for radon exposure. One end of the three-way cock was connected to the bottle containing blood through a needle and the other two ends to a 50-ml syringe and to the radium source or a vacuum pump. Such a set-up facilitates sequential evacuation and radon exposure without any loss of radon during exposure. The air in the bottle was partially evacuated using a vacuum pump and exactly 50 ml of a mixture containing radon gas and air was drawn into the syringe from the radium source. An exact volume of the gas was transferred to the bottle containing blood and another exact quantity from the same syringe was transferred to an evacuated Lucas cell and the activity counted immediately as well as after 3 h using an alpha counter. By varying the volume of radon gas taken in the syringe for each experiment different doses could be given to the blood cells. A fixed volume of radon air mixture was injected to the bottle containing blood sample and a smaller volume of the same radon air mixture into the Lucas cell. The second bottle containing blood was kept as control and sham exposed. Bottles were placed on a rocker platform inside a 37 ◦ C incubator for 3 h for uniform irradiation. Excess radon gas was released from the bottle after the exposure period by mild agitation and by merely opening the lid. 2.4. Estimation of radon concentration Radon activity was estimated using a Lucas cell coupled to an alpha counter. Concentration was converted to dose by using Marinelli’s formula. Details are given elsewhere [22]. Since radon emits 5.5 MeV radiation and assuming that the majority of exposure is due to radon rather than its progeny, doses were computed according to Marinelli formula taking into consideration energy, conversion factor, the mass and density of blood. Thus the activity obtained in kBq was converted to dose using Eq. (1). Dose(Gy) =

1.6 × 10−13 × 5.5 × kBq 11 × 10−3

(1)

2.5. Culture setup Whole blood culture was setup for the chromosome aberration assay and the cytokinesis block micronucleus assay using standard protocols [24]. Briefly, about 1 ml of whole blood was added to 9 ml of RPMI 1640 (HEPES modification, Sigma) containing 100 U/ml penicillin, 100 ␮g/ml streptomycin (Sigma) and 1 ml of fetal bovine serum (Sigma). For chromosome aberration assay, cultures were initiated by the addition of 5 ␮g/ml phytohemagglutinin (Sigma). Bromodeoxyuridine to a final concentration of 10 ␮M was added to the culture in order to differentiate first division cells. Cultures were incubated for 48 h in 5% CO2 atmosphere. At 46th hour 0.2 ml of colchicine working solution (0.04 ␮g/ml, Sigma) was added to arrest cells at metaphase. Cells were harvested at 48 h and subjected to a hypotonic treatment using 0.56% of KCl. Cells were washed and suspended in Carnoy’s fixative, cast on microscope slides, air-dried, stained with Giemsa and scored for aberrations.

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Table 1 Concentration and corresponding doses, dose-rates and chromosome aberration yield of radon gas. S. No.

Conc. of radon (kBq/m3 )

Dose (mGy)

Dose rate (mGy/min)

DC/cell

AF/cell

CR/cell

AR + DM/cell

CB/cell

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

0 122 239 265 359 89325 92648 100565 112341 216106 424132 424532 425314 438524 468725 1256354 1339843 1342915 1498132 1593210

0 0.01 0.019 0.02 0.03 7.1 7.4 8 9 17 33.93 33.96 34 35 37 101 107 107.4 120 127

0 0.000054 0.000106 0.000117 0.00016 0.0397 0.041 0.0447 0.0499 0.096 0.1885 0.1886 0.189 0.195 0.208 0.561 0.594 0.596 0.665 0.708

0.00043 0.0015 0.0023 0.0024 0.0028 0.0052 0.0057 0.0058 0.006 0.0074 0.0102 0.011 0.013 0.014 0.016 0.02 0.024 0.025 0.031 0.033

0 0.0015 0.0023 0.0048 0.0028 0.0029 0.0025 0.0029 0.0057 0.0092 0.0034 0.0037 0.0083 0.0096 0.013 0.012 0 0.025 0.021 0.0096

0 0 0.00115 0 0 0 0 0 0 0.00061 0 0 0 0.00069 0.0018 0.00066 0 0.0029 0.0023 0.00385

0.00026 0 0.00805 0.012 0 0.003 0.00063 0.00194 0 0.0018 0.0034 0.0012 0.0024 0.0021 0.0079 0.00066 0.0026 0.0139 0.028 0

0.00061 0 0 0.0024 0.0028 0.0012 0.0013 0 0 0.0006 0.003 0 0.0024 0 0.00061 0 0.0053 0.00096 0.0023 0

DC: dicentrics, AF: acentric fragment, CR: centric ring, AR: acentric ring, DM: double minute, and CB: chromatid break.

For CBMN assay, cytochalasin B (Sigma) at a concentration of 3 ␮g/ml was added to the culture at 44th hour and cells were harvested at 72nd hour after a hypotonic treatment with 0.45% of cold KCl. Cells were fixed in Carnoy’s, stained with Giemsa and scored.

2.6. Scoring of aberrations Metaphases were captured using an automated metaphase finder system (Metasystems, Germany). Individual first-division metaphases obtained from nonexposed as well as in vitro radon exposed samples were carefully analyzed and the aberrations were noted on to scoring sheets. Various types of chromosome aberrations such as dicentrics, centric and acentric rings, fragments, chromatid breaks and minutes were recorded. About 11518 metaphases were scored from control sample. For CBMN assay about thousand binucleate cells were scored manually for each dose as well as for control. Binucleate cells with one or more micronuclei, besides those containing nucleoplasmic bridges and nuclear buds were scored. Binucleate cells were scored if the two nuclei had intact nuclear membranes and were situated within the same cytoplasmic boundary. Cells with overlapping or touching nuclei were not scored. Nucleoplasmic bridges were scored if the bridge had the same staining pattern as that of the main nuclei. All cells were scored according to the strict criteria followed by Fenech [26]. 2.7. Statistical analysis Regression analysis was carried out using the statistical programme Origin 6.1. The distributions of aberrations were studied by the method described by Papworth and adopted by Savage [25]. This was done by the standard u-test using the formula: √ u = d −(N − 1)/ (var d), where N is the total number of cells scored, d is the coefficient of dispersion (N − 1) (2/Y), where Y is the mean number of observed aberrations, (2/Y) is the relative variance and var d is the variance of d given by 2(N − 1) (1−1/NY). This method makes use of the fact that the variances for a Poisson distribution equals the mean (Y).

Fig. 1. (A) Variation of dicentric yield at very low-dose-rates (0–0.096 mGy/min) of radon. (B) Variation of dicentric yield at very low-dose-rates (0.1885–0.708 mGy/min) of radon.

Fig. 2. Variation of centric ring yield with various dose of radon.

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Fig. 3. Variation of excess acentric fragment yield with various dose of radon. Plate 1. Metaphase spread with quadracentric chromosome and associated fragments.

3. Results 3.1. Chromosome aberration assay Details of concentrations, dose and dose-rates and various types of aberration yields are shown in Table 1. Aberrations such as dicentrics, excess acentric fragments, centric ring, acentric ring, double minutes and chromatid breaks were scored and their frequencies per cell were plotted against radon dose. Since dose-rates ranged significantly, they were classified into two low-dose-rate exposure groups, 0–0.096 mGy/min and 0.1885–0.708 mGy/min, respectively. The dicentric yields plotted according to these dose-rates are depicted in Fig. 1A and B. Regression analysis yielded a linear fit following the equations, Y = 0.00214D + 0.00037 and Y = 0.00424D + 0.0002 (R2 = 0.87, P < 0.0001, and R2 = 0.91, P < 0.0001) respectively. The frequency of centric rings also exhibited a dose–effect relationship (Fig. 2). As it was not possible to confidently distinguish acentric rings from double minutes, they were scored together as a single aberration and when plotted against dose did not exhibit a significant response. Excess acentric fragments and chromatid breaks were also observed in exposed

samples. The frequency of acentric fragments showed a significant dose–effect relationship (Fig. 3, Table 2). However, chromatid breaks did not show significant increase with dose. Metaphases with multiple aberrations, quadracentric chromosome, excess acentric fragments, acentric rings were observed and Plate 1 shows metaphase spread with quadracentric chromosomes. 3.2. Cytokinesis block micronucleus assay About 1000 binucleate cells were scored from each sample. Dose, dose-rates and aberration yields are shown in Table 3. In addition to scoring micronuclei in binucleate cells, other DNA damage events such as nucleoplasmic bridges and nuclear buds were also scored. Identification of MNi, NBUDS and NPBs was according to the criteria suggested by Fenech [26]. Frequencies of MNi, NBUDS and NPBs were plotted against radon dose. Binucleate cells with more than one MN were also scored. The frequency of MNi increased in a dose-dependent manner (Fig. 4, Table 4), whereas the frequencies of nucleoplasmic bridges and nuclear buds were not found to be sig-

Table 2 Distribution of acentric fragment yields in cells exposed to radon. Dose (mGy)

0 0.01 0.019 0.02 0.03 7.1 7.4 8 9 17 33.93 33.96 34 35 37 101 107 107.4 120 127

Cells scored

11518 1337 865 418 1442 1713 1580 1035 349 1632 294 809 834 1452 1647 1506 375 1039 872 519

No. of AF

0 2 2 2 4 5 4 3 2 15 1 3 7 14 22 18 0 26 18 5

AF distribution 0

1

2

3

11518 1335 863 416 1439 1708 1578 1033 347 1622 293 806 830 1443 1630 1492 375 1023 857 516

– 2 2 2 2 5 – 1 2 6 1 3 1 5 13 10 – 8 12 2

– – – – 1 – 2 1 – 3 – – 3 3 3 4 – 6 3 –

– – – – – – – – – 1 – – – 1 1 – – 2 – 1

AF: acentric fragment, PD: Poisson distribution, OD: over distribution.

AF yield ± S.E.

u

Distribution

0 0.0015 ± 0.0010 0.0023 ± 0.0016 0.0048 ± 0.0034 0.0028 ± 0.0014 0.0029 ± 0.0013 0.0025 ± 0.0013 0.0029 ± 0.0016 0.0057 ± 0.004 0.0092 ± 0.0024 0.0034 ± 0.0034 0.0037 ± 0.0021 0.0083 ± 0.0032 0.0096 ± 0.0026 0.013 ± 0.0028 0.012 ± 0.0028 0 0.025 ± 0.0049 0.02 ± 0.0048 0.0096 ± 0.0043

– −0.03 −0.03 −0.05 15.44 −0.08 32.40 18.53 −0.05 23.41 – −0.06 18.76 23.72 15.65 12.23 – 20.91 6.75 21.49

– PD PD PD OD PD OD OD PD OD – PD OD OD OD OD – OD OD OD

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Table 3 Concentration and corresponding radon doses, dose-rates and aberration yield obtained by CBMN assay. S. No.

Conc. of radon (kBq/m3 )

Dose (mGy)

Dose-rates (mGy/min)

Cells scored

MN yield ± S.E.

NPB yield ± S.E.

NBUDS ± S.E.

1 2 3 4 5 6 7 8 9 10 11 12 13 14

0 6499 14109 89325 92648 112341 202199 216106 438524 468725 503982 1092450 1256354 1498132

0 0.52 1.12 7.1 7.4 9 16 17 35 37 40.3 87.3 101 120

0 0.0028 0.0062 0.0397 0.041 0.0499 0.0888 0.096 0.195 0.208 0.222 0.485 0.561 0.665

1116 1002 1025 1001 1016 1004 467 1045 779 367 1006 1019 1013 1238

0.0215 ± 0.00043 0.004 ± 0.0019 0.043 ± 0.0065 0.04 ± 0.0063 0.029 ± 0.0054 0.016 ± 0.0039 0.023 ± 0.0071 0.046 ± 0.0066 0.020 ± 0.0051 0.065 ± 0.0133 0.035 ± 0.0058 0.054 ± 0.0073 0.060 ± 0.0077 0.092 ± 0.0086

0 0 0.001 ± 0.001 0.004 ± 0.0019 0.001 ± 0.001 0.001 ± 0.001 0.011 ± 0.0048 0.001 ± 0.001 0.0013 ± 0.0013 0.011 ± 0.0054 0.0019 ± 0.0014 0.0019 ± 0.0014 0.001 ± 0.001 0.013 ± 0.013

0 0 0 0.003 ± 0.0017 0 0 0.0042 ± 0.003 0.00095 ± 0.00095 0.0013 ± 0.0013 0.0054 ± 0.004 0 0 0 0.0097 ± 0.0028

MNi: micronuclei, NPB: nucleoplasmic bridges, and NBUD: nuclear bud.

Fig. 4. Variation of micronuclei yield with various dose of radon. Plate 2. Binucleate cell with four micronuclei.

nificantly elevated with increase in radon dose (data not shown). Plate 2 shows binucleate cells with four micronuclei. Plate 3 shows binucleate cells with NPB accompanied by a micronucleus. Plates 4 and 5 show binucleate cell with a nuclear bud and binucleate cell with nucleoplasmic bridge, MN and nuclear bud, respectively. There was a significant correlation between nucloeplasmic bridges and dicentric plus centric rings. Regression analysis yielded a slope of 0.2865 (P < 0.0339), considered to be significant (Fig. 7). When plotted individually against the frequency of

dicentrics or centric ring, the correlation was also very significant (Figs. 5 and 6). Excess acentric fragments plotted against MNi showed a significant correlation (Fig. 8). MNi induced by ionizing radiation or clastogens mostly arise from acentric chromosome fragments while those induced by aneuploidogens are from whole chromosomes [27–29].

Table 4 Distribution of micronuclei yields in cell exposed to radon. Dose (mGy)

Cells scored

No. of MN

0 0.52 1.12 7.1 7.4 9 16 17 35 37 40.3 87.3 101 120

1116 1002 1025 1001 1016 1004 467 1045 779 367 1006 1019 1013 1238

24 4 44 40 30 16 11 48 16 24 35 55 61 114

MNi distribution 0

1

2

3

4

5

6

7

8

1093 998 987 975 997 990 459 1005 769 350 976 977 965 1150

22 4 33 21 15 12 4 34 7 12 25 34 39 66

1 – 4 2 4 2 3 4 2 4 5 4 5 20

– – 1 1 – – 1 2 – – – 3 4 1

– – – 1 – – – – – 1 – 1 – –

– – – – – – – – 1 – – – – 1

– – – – – – – – – – – – – –

– – – – 1 – – – – – – – – –

– – – 1 – – – – – – – – – –

MNi yield ± S.E.

u

Distribution

0.0215 ± 0.00043 0.004 ± 0.0019 0.043 ± 0.0065 0.04 ± 0.0063 0.029 ± 0.0054 0.016 ± 0.0039 0.023 ± 0.0071 0.046 ± 0.0066 0.020 ± 0.0051 0.065 ± 0.0133 0.035 ± 0.0058 0.054 ± 0.0073 0.060 ± 0.0077 0.092 ± 0.0086

1.51 −0.08 6.33 43.32 37.57 5.44 14.29 8.59 30.20 10.68 5.74 14.54 11.31 12.19

PD PD OD OD OD OD OD OD OD OD OD OD OD OD

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Fig. 5. Correlation between nucleoplasmic bridges and dicentrics. Plate 3. Binucleate cell with nucleoplasmic bridge and one MN.

4. Discussion

Plate 4. Binucleate cell with nuclear bud.

Plate 5. Binucleate cell with nucleoplasmic bridge, MN and bud.

Studies at molecular, cellular, animal and human levels have been carried out to understand the carcinogenic effects of radon, which emits high LET alpha radiation during its decay. The double strand breaks induced by alpha particles are highly localized cluster damages and repair more slowly when compared to damages induced by low LET radiation [30]. Quantitative cytogenetic assays involving in vitro and in vivo structural chromosome aberration analysis in human peripheral blood lymphocytes have been carried out to study the effect of radon gas. Various studies proved that dicentric assay is the most radiation specific in the moderate to high dose range [31]. In the present study cytogenetic effects of radon and its daughter progenies using two well known biomarkers–chromosome aberrations and cytokinesis block micronucleus were analysed and compared. In our study there was a significant increase in dicentric and centric ring frequencies with increase in radon dose (Figs. 1 and 2). Reports indicate statistically significant increase of chromosome aberrations in Uranium miners occupationally exposed to 222 Rn compared to controls [32–34]. Bauchinger et al. have shown an increase in dicentric and ring chromosomes in blood lymphocytes of persons living in houses with very high radon concentrations [35].

Fig. 6. Correlation between centric ring and nucleoplasmic bridge yield.

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Fig. 7. Correlation between nucleoplasmic bridges and dicentrics + centric rings.

Fig. 8. Correlation between micronuclei and acentric fragments.

The incidence of excess acentric fragments was found to increase in a dose dependent manner. Similar results were observed by others [36,37]. On comparing Table 2, Figs. 3 and 4, it can been seen that the AF frequency and MNi increases with dose while its distribution is non-Poisson indicating clustered damage and hence a signature of high-LET radiation. A similar trend was also observed for the incidence and distribution of dicentrics [22]. On comparing previous data on MNi induction by radon it is seen that most reports deal with doses greater than 100 mGy while in the present study doses ranged between 0.52 and 120 mGy. Hence it is difficult to compare results per unit of damage. At the few doses overlapping with this study, the MN yield is comparable with that observed by Khan et al. [20] and the DC yield comparable with that of Pohl-Ruling et al. [38]. On comparing the dose–response relationships, the alpha coefficient obtained for MN yield by Khan et al. is about 0.76 greater than that obtained in the present study. This could be due to a lower concentration of Cytochalasin-B (3 ␮g/ml) used in the present study that could have resulted in an underestimate of MNi. Publications with alpha-emitters suggest there is little dose-rate effects while some suggest the existence of an inverse dose-rate effect [39–41]. In the present study the alpha coefficient of the dose–response relationship is found to be lower at the lower doserate by about a factor of two (Fig. 1A and B) and there appears to be absence of an inverse dose-rate effect. Nevertheless it is difficult

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to confirm these effects in the absence of certain dose points in the 0.04–7 mGy range. The CBMN assay has been proved effective in the study of chromosome damage induced by ionizing radiation [42–44] and a recent report shows that the assay can be used as a biological dosemeter for targeted alpha therapy [45]. In our study there was a significant increase in MNi yield with increase in radon dose. The background frequency of MNi yield in the present study was found to be 0.0215. High background frequency of micronuclei was also observed by others [46]. Besides, MNi and AF correlate well suggesting the bulk of MNi produced are likely to be from AFs. MNi are thought to be manifestations of lagging chromosomes or chromosome fragments and in the present study the frequency of excess acentric fragments correlated well with that of MNi (P < 0.00496) in radon exposed samples. The scoring of nucleoplasmic bridges and nuclear buds is a recent trend in the CBMN assay. It has been suggested that the incidence of nucleoplasmic bridges might be a sensitive indicator of clastogenic DNA damage as it has shown a lower background frequency [47]. The rationale being that nucleoplasmic bridges, envisaged to arise from dicentric and centric ring chromosomes are likely to be more specific to ionizing radiation. A detailed picture of the probable mechanism underlying the formation of NBPs has been outlined by Fenech [21]. In a recent report occupational workers in a nuclear industry were found to exhibit nucleoplasmic bridges in CBMN assay [48]. The analysis of NPBs as a biomarker of DNA damage in human WIL2-NS cells treated with hydrogen peroxide, superoxide or after co-incubation with activated human neutrophils has been recently validated [49]. In the present study, nucleoplasmic bridges with and without micronuclei were observed in radon exposed samples. BN cells containing nucleoplasmic bridges with MN are thought to be the result of dicentric chromosomes and its associated fragment. The apparent absence of a MN in cells exhibiting nucleoplasmic bridges could be due to masking effect of the macronuclei or telomere end fusion [21]. Radon exposed BN cells also exhibited nuclear buds and are thought to be formed as a result of gene amplification. Double minutes and nuclear buds are thought to be formed as a result of gene amplification [26,50,51]. In the present study as it was difficult to truly differentiate acentric rings from DMs, it was not possible to correlate DMs and gene amplification. Complex aberrations, characteristic of high LET radiation were seen in metaphase spreads exposed to radon. In CBMN assay also we encountered “complex BN cell” with micronuclei, nucleoplasmic bridge and nuclear bud like structure as the one shown in Plate 5. Alpha particles produced by radon and its progenies cause a significant amount of DNA damage by the “direct effect” and are more clustered in the scale of DNA and chromatin. The damage thus produced will be difficult to repair. It has also been suggested that separate double strand breaks produced by the same track can interact with each other because of the higher order chromatin structure [2] and may lead to the production of excess acentric fragments [52] and very large deletions up to about 200 kb [53]. Repair of these damages will be more difficult and slower [54]. Till date there have been very few reports on the incidence of NPBs and NBs in human lymphocytes using the CBMN assay. Thomas et al. [47] reported a dose-dependent increase in MN, NPBs, AFs, DC and rings in cells exposed to high doses of gamma radiation. In the present study we observed an increase in MNi, AFs, DC and rings in cells exposed to alpha radiation at low doses (10–100 mGy). This study clearly shows that very low doses of alpha radiation are capable of inducing MN, NPBs and NBUDS in human lymphocytes. The probable role of bystander effects [49] needs to be explored by a more systematic study controlling parameters such as dose-

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