Medical

Communication

Biosci. Biotech. Res. Comm. 10(1): 56-62 (2017)

Low levels of ionizing radiation exposure and

cytogenetic effects in radiopharmacists

T Soltanpour Gharibdousty

, F Zakeri

2,3

, V Changizi

*, MR Rajabpour

and MR Farshidpour

Department of Technology of Radiology and Radiotherapy, Allied Medical Sciences School, Tehran University

of Medical Sciences, Tehran, Iran

Nuclear Science and Technology Research Institute, Tehran, Iran

Iran Nuclear Regulatory Authority, Tehran, Iran

ABSTRACT

Radiopharmaceuticals are unique medicinal formulations containing radioisotopes which are used in major clini-

cal areas for diagnosis and/or therapy. The aim of this study was to assess occupationally induced DNA damage in

workers of a radiopharmaceutical facility who are at risk of exposure to low levels of external ionizing radiation

and internal contamination. Cytokinesis-block micronucleus (CBMN) assay used as an important biological indicator

of ionizing radiation exposure in the peripheral blood lymphocytes of 35 exposed workers compared with 35 con-

trols matched for gender, age and smoking habits. Occupational dosimetry records were collected over the last year

(ranged from 1.05 to 30.73 mSv) and last 5 years exposure (ranged from 2.56 to 70.24 mSv). The results showed a

2.5 fold increase in the chromosomal damage leading to micronucleated lymphocytes in the workers of the radiop-

harmaceutical facility compared to the controls (25.82±8.67 vs. 10.52±6.83 micronuclei per 1000 binucleated cells,

p < 0.0005). The nuclear division index as a parameter of cytostasis, in the workers was signi cantly lower than

that in the controls. The mean frequency of nucleoplasmic bridges was higher in the radiation workers compared to

the controls without statistical signi cant difference (p > 0.05). Taking all the confounding factors into account, no

obvious trend of increased micronuclei as a function of either duration of employment, exposed dose, smoking or

age was observed. The present study showed that occupationally exposed individuals have higher frequencies of DNA

damage, despite the very low levels of ionizing radiation exposure.

KEY WORDS: OCCUPATIONAL EXPOSURE, RADIOPHARMACIST, MICRONUCLEI, HUMAN LYMPHOCYTE

ARTICLE INFORMATION:

*Corresponding Author: changizi@sina.tums.ac.ir

Received 12

Jan, 2017

Accepted after revision 22

March, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

T Soltanpour Gharibdousty et al.

INTRODUCTION

The development of nuclear energy and the growing use

of ionizing radiation in medical practice have created

deep concern regarding the long-term effects of low-dose

radiation on humans (Mozdarani and Samavat, 1996).

Positron emission tomography (PET) is an indispensable

imaging modality for the evaluation and staging of can-

cer patients by using radiotracers or radiopharmaceuti-

cals, labeled with suitable positron emitting radioisotopes

and necessitate special consideration in manufacturing

and working knowledge of radiation safety practices is

also essential. Workers in radiopharmaceutical facilities

are involved in different sections of Radionuclide produc-

tion and Radiopharmaceutical preparation and are at risk

of external radiation exposure and internal contamina-

tion during their work, (IAEA, 2012).

Generally, monitoring of individuals occupationally

exposed to ionizing radiation consists of regular  lm

dosimetric control and periodic health examination

(Zakeri et al., 2003) However, personal dosimetry may

underestimate the real exposure, not only because of the

detection threshold of dosimeters but also because of

improper wearing (Zakeri and Hirobe, 2010, Sari-Min-

odier et al., 2007). Cytogenetic studies as speci c bio-

logical parameters provide additional information which

complements physical dosimetry and enables better

evaluation of radiation exposure.The cytokinesis-block

micronucleus (CBMN) assay are widely used as end

points in testing for mutagens and carcinogens (Pres-

ton, 1984, Sorsa, 1990) and micronuclei (MN) induction

re ects clastogenic and/or aneugenic events (Sari-Min-

odier et al., 2007, Zakeri and Hirobe, 2010).

MN observed in peripheral blood lymphocytes (PBL),

in the  rst interphase after cell division, are small extra-

nuclear bodies resulting from chromosome breaks or

whole chromosomes lagging behind during anaphase.

These cells can be identi ed as binucleated (BN) cells by

addition of the cytoplasmic division inhibitor cytocha-

lasin B during cell culture. Ionizing radiation is a strong

clastogenic agent, and thus a potent inducer of MN (Vral

et al., 2011).

The CBMN assay has been extensively used and vali-

dated as an appropriate bio dosimetry tool to evaluate

in vivo radiation exposure of occupational, medical and

accidentally exposed individuals and to assess in vitro

radiosensitivity and cancer susceptibility (Sari-Minodier

et al., 2007, Dias et al., 2007, Thierens and Vral, 2009).

Many studies have shown that the number of radiation-

induced MN strongly correlated with dose and quality of

radiation. Most studies reported that MN in lymphocytes

was more frequent in radiation workers than in the con-

trols (Bonassi et al., 1997, Chung et al., 1996 and Vral

et al., 2011).

In this study we examined the frequency of MN in

35 radiopharmacists occupationally exposed to ioniz-

ing radiation and 35 sex- and age-matched controls.

This is important as signi cantly elevated levels of such

aberrations may be found in peripheral blood lympho-

cytes of radiation workers who are exposed within the

occupational limits recommended by the International

Commission on Radiation Protection (ICRP and 1991.).

Parameters of cell cycle progression, other biomarkers of

DNA damage such as nucleoplasmic bridges and effects

of confounding factors on MN induction have been also

investigated.

MATERIAL AND METHODS

STUDY POPULATION

All the study population was male. The exposed group

comprised 35 radiopharmacists involved in prepara-

tion and production of radiopharmaceuticals. Their age

ranged from 27 to 55 years (mean of 37.59±7.22 years)

with mean employment time of 14.35±8.71 years. The

test group was routinely monitored with  lm badges

every 2 months. Their exposure ranged between 1.05

and 30.73 mSv (average of 6.59±5.83 mSv/y) during the

previous year and from 2.56 to 70.24 mSv with a mean

value of 17.89±15.04 mSv during the last 5 years.

The reference control group consisted of 35 individu-

als, who had never been occupationally exposed to ion-

izing radiation or to the known carcinogenic chemicals.

The controls matched the exposed subjects in age, sex

and smoking habits.

All subjects in this study were questioned in detail to

learn if they were systemically healthy. The question-

naire also included information about smoking habits,

medical history, drug intake and diagnostic medical irra-

diation for all individuals. For the exposed subjects, the

type, frequency and duration of occupational exposure

to ionizing radiation, radiation protection measures,

conditions of dosimeter wearing and work-related expo-

sure to other hazardous agents were questioned. All the

subjects were healthy individuals without current infec-

tions and medications in the last 6 months and no gen-

eral or dental X-ray in the last 6 months.

The study was approved by the national ethical com-

mittee. Informed consent was obtained from each person

and the study protocol conformed to the ethical guide-

lines of the World Medical Association (Declaration of

Helsinki).

Cytokinesis-block micronucleus (CBMN) assay

Peripheral blood samples (3ml) were collected into steri-

lized lithium heparin tubes by venipuncture from each

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS LOW LEVELS OF IONIZING RADIATION EXPOSURE AND CYTOGENETIC EFFECTS 57

T Soltanpour Gharibdousty et al.

FIGURE 1. Various binucleated cells (BN) observed in the cytokinesis-block

micronucleus assay; a) a normal BN, b) a BN with a micronucleous (MN) and a

nuclear bud, c) a BN with 2 MN and one nuclear bud, d) a BN with 2 MN and

one nucleoplasmic bridge, e)Two BN with one and 2 MN, f) a BN with 3 MN.

donor and all samples were coded. Whole blood cultures

were created by mixing 0.5 ml of whole blood with 4.5

ml of RPMI 1640 medium consisting of Ham’s F10 sup-

plemented with 20% fetal calf serum (FCS), 100 U/ml

penicillin, 100 mg/ml streptomycin, 1.0% l-glutamine

and 1.0% phytohemagglutinin (PHA) for mitogenic

stimulation (all materials purchased from Gibco). The

cultures were incubated at 37 0C for 72 h. Cytochalasin

B (6 mg/ml) was added 44 h after culture initiation. The

cells were collected by centrifugation, and treated with

a mild hypotonic solution containing 0.075 M KCl for 3

min. After centrifugation and removal of the superna-

tant, the cells were  xed with a fresh mixture of metha-

nol: acetic acid (3:1). Centrifugation and re-suspension

were carried out three times and the cells were then

dropped onto clean slides for detection of micronuclei

by conventional staining with 5% Giemsa (Zakeri and

Hirobe, 2010).

MN was scored in binucleated cytokinesis-blocked

cells using 400 X magni cation. The frequencies of

MN and nucleoplasmic bridges (NPBs) were determined

at the same time in binucleated cells according to the

standard criteria described by Fenech (Fenech, 1993)

were followed for identi cation of binucleated lympho-

cytes and MN. Figure 1 shows the various DNA damages

observed in the cytokinesis-block micronucleus assay.

The One thousand binucleated cells were examined for

each sample. Furthermore, Cell cycle parameters were

evaluated by classifying 1000 cells according to the

number of nuclei. The nuclear division index (NDI) was

calculated following the formula: NDI = (M1 + 2M2 +

3M3 + 4M4) /n, where M1–M4 indicate the number of

cells with 1–4 nuclei and n indicates the total number of

scored cells (Rastkhah et al., 2016)ÿÿÿÿÿþ»n4ÿÿ.

STATISTICAL EVALUATION

The frequency of MN and NDI value in the lymphocytes

of the workers and controls were compared using Stu-

dent’s t test. The in uence of age, duration of employ-

ment and physical doses was tested by linear regression

analysis and correlation test using SPSS, version 21 sta-

tistical program. The level of statistical signi cance was

set at p<0.05.

RESULTS

Demographic characteristics of control and exposed

workers (r adiopharmacists) are presented in Table

1. There was no difference in age and smoking sta-

tus distributions between control and exposed group

(Table 1).

Results of the MN, NPB and NDI frequencies have

been shown in Table 2. The mean frequency of MN/1000

binucleated cells of radiation workers were signi -

cantly higher than in the control group (25.82±8.67

vs. 10.52±6.83, p <0.0005, student’s t-test). The mean

frequency of NPBs was higher in the radiation work-

ers compared to the controls but there was no statisti-

cally signi cant difference (p > 0.05). The mean value

58 LOW LEVELS OF IONIZING RADIATION EXPOSURE AND CYTOGENETIC EFFECTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

T Soltanpour Gharibdousty et al.

Table 1. Demographic characteristics of the radiopharmacists and the control group. Data are

expressed as mean values with standard deviation.

Groups Radiopharmacists Controls p value

Mean age, years± S.D.

(range)

(no.)

37.59±7.22

(27-55)

(35)

36.98±7.44

(25-55)

(35)

0.59

Smoking habits, no. (%)

Yes

14(40.0%) 10(28.75%) 0.32

No 21(60.0%) 25(71.25%)

Mean time exposure, years± S.D. 14.35±8.71 - -

Mean last year exposure mSv± SD

(range)

6.59±5.83

(1.05 – 30.73)

Mean last  ve years exposure mSv± SD

(range)

17.89±15.04

(2.56 -70.24)

S.D: standard deviation

Table 2. Frequency of MN and NDI in lymphocytes of the radiopharmacists and the

control group

Groups Radiopharmacists Control p value

Mean MN frequency(‰) ± SD

(range)

(no.)

25.82±8.67

(11-48)

(35)

10.52±6.83

(2-23)

(35)

<0.0005a

Mean NDI frequency(‰) ± SD

(no.)

1.76 ±.02

(35)

1.84±.03

(35)

<0.0005a

Mean NPB frequency(‰) ± SD 1.02±0.47 0.85±0.37 NS

Mean MN frequency(‰) ± SD

Smokers (no.)

Non-smokers (no.)

27.46±9.01(14)

24.13±7.54(21)

p=0.35

11.51±6.89 (10)

9.54± 4.21(25)

p=0.44

MN: micronuclei, NDI: Nuclear division index, NPB: Nucleoplasmic bridge

a :obtained by Student ’s t-test

S.D: standard deviation

NS: not signi cant

of NDI; a parameter of cytostasis, for radiation workers

and controls were 1.76±.02 and 1.84±.03, respectively.

Although both are in the normal range, however, the

NDI frequency in the control group was signi cantly

higher than that in the workers (p < 0.0005, student’s

t-test).

The regression analysis showed no signi cant cor-

relation emerged between age (=0.06, p=0.7), time

exposure (=0.02, p=0.9), dose in the last year (=0.01,

p=0.9) and dose in the last 5 years (=0.10, p=0.5) with

the frequencies of the MN/1000 BN cells in the exposed

workers. There were no signi cant differences between

the mean MN/1000 BN cells in the smoker and non-

smoker exposed workers (26.12± 10.32 vs., 22.0± 6.22,

p=0.20) and between smoker and non-smoker controls

(11.31± 7.51 vs., 9.70± 4.20, p=0.48) analyzed by Stu-

dent’s t-test.

DISCUSSION

In the present study, the CBMN assay was used to evalu-

ate chromosomal damage in peripheral blood lympho-

cytes of 35 radiopharmacists working in a radiophar-

maceutical facility involving in the preparation and

production of radionuclides and radiopharmaceuti-

cals in comparison with their sex- and age-matched

controls.

Although the level of radiation dose received occu-

pationally by workers was mostly below the accepted

annual limit of 20 mSv/year, the frequencies of MN were

signi cantly higher in the workers than in the controls

(25.82±8.67 vs. 10.52±6.83 per 1000 binucleated cells,

p <0.0005). A relatively high frequency of MN forma-

tion in lymphocytes of radiation workers compared with

non-exposed individuals might be due to an accumula-

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS LOW LEVELS OF IONIZING RADIATION EXPOSURE AND CYTOGENETIC EFFECTS 59

T Soltanpour Gharibdousty et al.

tion of initial DNA damage in people exposed to chronic

doses of radiation.

Many studies reported signi cantly higher MN rates

in exposed population than in controls (Sari-Minodier

et al., 2007, Zakeri and Hirobe, 2010). Maluf et al (Maluf

et al., 2001) also reported increased frequency of MN and

dicentric bridges in the lymphocytes of medical workers

exposed to X-rays. These results are also in agreement

with those who showed a signi cant higher MN val-

ues in interventional cardiologists when compared with

clinical cardiologists working outside the catheterization

laboratory (Andreassi et al., 2005).

Indeed age, gender and smoking habit are the most

important demographic variables impacting on the MN

index (Fenech, 1998). The age-related decline in the ef -

ciency of the repair processes and the accumulation of

mutations may lead to increased levels of DNA damage,

which is observed by a higher frequency of chromo-

somal aberrations (Wojda et al., 2007).

In addition to age, gender-related changes in the level

of MN frequency have been observed in many studies.

Effect of gender on MN formation has been described for

X chromosome micronucleation as being prevalent in

females (Mateuca et al., 2006). According to Fenech et al

(Fenech et al., 1994), an additional mechanism, possibly

the loss of the X chromosome, accounts for the higher

MN frequency in female. However, since the surveyed

groups in this study were all men and the mean age was

relatively the same in both groups, these effects have not

been observed.

Although cytokinetic and cytostatic effects have been

detected in heavy and moderate smokers (Calderon-

Ezquerro et al., 2007), smoking habits may or may not

affect the genotoxic effect of chronic radiation exposure

(Maffei et al., 2002, Sari-Minodier et al., 2007, Hadjide-

kova et al., 2003). Bonassi et al (Bonassi et al., 2003)

evaluated the impact of smoking on MN frequency using

pooled re-analysis of 24 databases from the Human pro-

ject. Our study indicated that smokers do not experience

an overall increase in the MN frequency compared with

non-smokers.

In this study, as reported by others, no association

between chromosome damage and absorbed dose was

found. The lack of a dose–effect relationship between

chromosome damage and chronic exposure to low lev-

els of ionizing radiation could be attributed to various

factors, which can be explained by the fact that during

chronic exposure part of the chromosomal damage can

be eliminated in vivo by the death of lymphocytes. This

can be attributed to proliferation rate of lymphocytes,

inter-individual variation, elimination of aberrant cells,

and lifespan of lymphocytes (Kubelka et al., 1992).

In this study the frequencies of NPBs were higher in

the exposed workers than in the controls without sta-

tistically signi cant difference (p > 0.05). NPBs origi-

nate from dicentric chromosomes that may be caused by

misrepair of double strand DNA breaks or telomere end

fusions. NPBs occur when centromeres of dicentric chro-

mosomes are pulled to opposite poles of the cell ana-

phase (Fenech, 2007). However, previous studies have

shown that NPB is not radiation speci c because it can

also be induced by reactive oxygen species (Umegaki

and Fenech, 2000).

NDI may be used to de ne cell cycle progression of

the lymphocytes after mitogenic stimulation and it is a

useful research tool for understanding the cell cycling

kinetics of the cultures specially after radiation expo-

sure (Rastkhah et al., 2016)ÿÿÿÿÿ-Fm4ÿÿ. Our results

showed a signi cant decrease in the NDI values of radi-

ation workers compared with non-exposed individuals

(p> 0.0005), that could be due to the chronic low dose

exposure. Signi cant decrease in the pooled NDI values

with increasing radiation doses was also observed by

other studies (Antunes et al., 2014, Pejchal et al., 2011,

IAEA, 2001.).

The increased micronuclei frequencies found in

exposed workers indicate potential genetic hazards that

may play critical role in radiation-induced carcinogene-

sis and genetic diseases in long term (Zakeri and Hirobe,

2010). It has been reported that the processes of pre-

paring and dispensing therapeutic radiopharmaceuticals

have a greater potential to expose operators to radia-

tion than do procedures for diagnosis. These operations

should therefore be performed in a controlled area with

entry restricted to essential staff only and careful con-

sideration should be given to the amount of shielding

required and to the measures to be taken to avoid radia-

tion exposure from internal contamination (AGENCY,

2011).

In radiopharmaceutical facilities, the main precau-

tions required in dealing with external irradiation will

depend on the physical characteristics of the radiation

emitted, the total activity and the physical half-life of

the radionuclide. Contamination from unsealed radioac-

tive substances may produce a further external radia-

tion hazard (Radiation and Protection, 2008).Now, it is

becoming increasingly clear that long-term radiation-

induced cancer risk can be dramatically minimized by

effective implementation of good practice of radiation

protection in radiopharmaceutical facilities and ‘respon-

sibility is on all workers and professional staff to mini-

mize the radiation exposure to themselves (Shadley

et al., 1987).

Therefore, our  ndings suggest that radiation work-

ers should be aware of the necessity of applying radia-

tion protection principles (justi cation, optimization

and limitation) and con rm that the regulatory body

shall establish and enforce requirements for the protec-

60 LOW LEVELS OF IONIZING RADIATION EXPOSURE AND CYTOGENETIC EFFECTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

T Soltanpour Gharibdousty et al.

tion of workers in existing exposure situations and sup-

port services such as education and training, technical

services, regular maintenance of equipment and quality

control program. Furthermore, evaluation and follow-up

of occupational doses should be considered as an impor-

tant part of quality assurance programs. In this regard,

cytogenetic monitoring, specially by using CBMN assay

is a valuable tool versus fIlm dosimetry following low-

dose radiation exposure and for risk assessment of per-

sonnel believed to be exposed to radiation.

ACKNOWLEDGMENT

This study has been supported by Tehran University of

Medical Sciences. Grant number:27610

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