A cytogenetic biomonitoring of industrial radiographers occupationally exposed to low levels of ionizing radiation by using cbmn assay

Abstract

Industrial radiography is the process of using either gamma-emitting radionuclide sources or X-ray machines to examine the safety of industrial materials. The average annual effective dose in industrial radiography is one of the highest among radiation workers. The aim of this study was to investigate the cytogenetic effects of ionizing radiation in the peripheral blood lymphocytes of 60 industrial radiographers and 40 non-exposed individuals as the control group by using cytokinesis-block micronucleus (CBMN) assay. Totally, the frequencies of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) were significantly higher in the industrial radiographers than in the controls (p = 0.000). The mean MN frequency per 1000 binucleated cells in the industrial radiographers with last 5-y radiation dose of >100 mSv was significantly higher than those with ≤100 mSv (34.81 ± 12.7‰ vs. 26.33 ± 7.94‰, p = 0.024). The effect of age was observed in the control group and subjects with the age of >30 y showed significantly higher MN frequency compared with the subjects with the age of ≤30 y (9.45 ± 3.71‰ vs. 6.81 ± 3.05‰, p = 0.02). No obvious trend of increased MN as a function of either duration of employment or age or smoking status was observed in the industrial radiographers. The results show the increased levels of cytogenetic damages in the industrial radiographers. Even the workers exposed to the permissible doses are subjected to elevated frequencies of DNA damages. These findings confirm the importance of cytogenetic biomonitoring program beside physical dosimetry, surveying radiation safety of equipment and periodic training of workers for improvement of safety and radiation protection.

INTRODUCTION

The exposure of human being to ionizing radiation from natural sources is a continuing and unavoidable feature of life on the earth⁽¹⁾. For most individuals such as radiation workers, this exposure exceeds from all man-made sources⁽¹⁾. These exposure as a result of occupational activities incurred by workers in industry, medical and research using radiation or radioactive substances⁽²⁾.

Industrial radiography is a usual procedure for producing a radiograph by using either gamma-emitting radionuclide sources or X-ray machines to examine the safety of industrial materials⁽³⁾. The average annual effective dose in industrial radiography is one of the highest among all practices and the rate of accidental exposures in this practice is high^{(4, 5)}.

Ionizing radiation induces the formation of acentric chromosome fragments and to a small extent malsegregation of whole chromosomes⁽⁶⁾. Acentric chromosome fragments and whole chromosomes that are unable to connect with the spindle lag behind at anaphase are not included in the main daughter nuclei^(6,7). A lagging chromosome fragment or whole chromosome forms into a small separate nucleus is called micronucleus (MN)⁽⁶⁾.

The cytokinesis-block micronucleus (CBMN) assay in human lymphocytes has become one of the most widely used methods for measuring structural and numerical chromosomal changes in human cells in vitro and in vivo⁽⁸⁾. The CBMN assay is a reliable test to assess radiation-induced chromosome damage and is a valuable biomarker in many biomonitoring studies among human populations occupationally or environmentally exposed to ionizing radiation^(9,10). MN also are an intermediate endpoint of carcinogenesis and a long-term predictor of cancer^{(11, 12)}.

Most of the cytogenetic studies of individuals occupationally exposed to the low doses of radiation declared a significantly higher level of chromosome aberration and MN than control^(13–16). Cytogenetic studies by using fluorescent in situ hybridization (FISH) with pancentromeric probe on MN showed a significantly higher frequency of micronuclei without centromere (C⁻MN) in the radiation workers^{(3, 9, 17, 18)} that show the clastogenic effect of ionizing radiation.

A previous study on industrial radiographers in Iran showed that the main causes of overexposures in this group were difficult working conditions and ignoring safety principles while accidents or device failures were a minor contribution⁽⁵⁾. Therefore, in order to provide additional information that complements physical dosimetry and for better evaluation of radiation health effects we performed a cytogenetic analysis in peripheral blood lymphocytes of industrial radiographers using the CBMN cytogenetic assay. Apart from the evaluation of MN, other relevant biomarkers of nucleoplasmic bridges (NPBs), nuclear buds (NBUDs) and proportion of dividing cells (parameter of cytostasis) have been assessed. Furthermore, the effects of confounding factors such as age, dose, smoking status and duration of exposure on DNA damages were evaluated in industrial radiographers and control.

MATERIALS AND METHODS

Subject

The study was performed in two groups of industrial radiographers in Iran. Group A: 35 overexposed industrial radiographers (mean age of 34.03 ± 5.63 y) who had dosimetrically recorded exposure doses higher than annual limit of 20 mSv. As recommended by ICRP (International Commission on Radiological Protection) the annual dose limit is 20 mSv ,the maximum allowable in any single year is 50 mSv with no more than 100 mSv in 5 y⁽¹⁹⁾. Group B: 25 industrial radiographers (mean age of 38.64 ± 5.9 y) who had no dosimetrically recorded exposure dose higher than 20 mSv in a year and not more than 50 mSv in last 5 y. All the study population was male. The result compared with 40 non-exposed male employees working mainly in administrative offices in the capital city of Tehran and matched by age, sex and lifestyle. All the industrial radiographers were using Iridium-192 source for non-destructive testing. The official personal dosimetry records, based on bimonthly thermoluminescent dosimetry (TLD). The dosimetric data of last 1 y and last 5 y exposure were collected for each radiographer.

All subjects were questioned in detail to learn whether they were systemically healthy. No subjects were exposed to any chemical exposure, medical irradiation or had alcohol intake. Individuals with personal medical history of disease, cancer and drug intake during last 6 months before this study were excluded from both groups. All participants’ rights were protected and written informed consent was obtained from all donors.

CBMN assay

The MN were prepared in cytokinesis-blocked cells using cytochalasin B (Cyt-B, Sigma) according to Fenech and Morley⁽²⁰⁾. Two separate cultures from each sample were set up by mixing 0.5 ml of sodium-heparinized blood with 4.5 ml of RPMI 1640 medium (Gibco). The cultures were incubated at 37°C for 72 h. Cytochalasin B (6 µg ml⁻¹) (Sigma) was added 44 h after culture initiation. Cells were then harvested and fixed with a fresh mixture of methanol: acetic acid (6:1). Cells were stained with 5% Gimsa (Merck). For each subject, a total of 1000 binucleated cells with well-preserved cytoplasm (500 per replicate) were scored blindly by the same reader under an optical microscope. Scoring was done according to the criteria of Fenech⁽²¹⁾. Also, to evaluate cell-cycle parameters 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. The frequencies of other cytogenetic biomarkers such as NPBs and NBUDs scored in 1000 binucleated cells for each subject according to the criteria of Fenech⁽²²⁾.

Statistical analysis

To evaluate the differences between the industrial radiographers and controls in terms of general characteristics, ANOVA or χ2- tests were used. The normality of dependent variables (MN, NPB, NBUD and NDI) distributions were evaluated by Kolmogorov–Smirnov test and logarithmic transformation of variables. Differences in the frequencies of genotoxic biomarkers including MN, NPB, NBUD and NDI between industrial radiographers and control groups were analyzed using ANOVA. ANOVA was performed to evaluate the effects of age, smoking status and duration of occupational exposure and multiple regression was performed to evaluate the effects of radiation exposure as independent variables on the frequencies of MN as a dependent. Data were analyzed with IBM SPSS Statistics for Windows package, version 22. Data are expressed as mean (±S.D.) and statistical significance was set at p < 0.05.

Results

Table 1 shows demographic characteristics of control and industrial radiographers. The control and exposed subjects were all male and did not significantly differ in the age and smoking habits. As shown in Table 2, MN frequencies were significantly (p = 0.000) higher among both groups of workers compared with the controls. And a borderline difference of MN frequency was observed between two groups of industrial radiographers (p = 0.049).

Table 1.

General characteristics of the industrial radiographers and control.

	Industrial radiographers		Control	p-value
	A	B
Total, n (%)	35 (35)	25 (25)	40 (40)
Mean age, y ± S.D.	34.03 ± 5.63	38.64 ± 5.9	35.11 ± 3.09	0.44
Range	23–46	29.51	23–48
<30 y old, n (%)	22 (62.8)	9 (36)	16 (40)
≥30 y old	13 (34.2)	16 (64)	24 (60)
Smoking status, n (%)				0.22
Current	12 (34.3)	9 (16)	4 (10)
Never	23(65.7)	21 (84)	36 (90)
Mean duration of exposure, y ± S.D.	8.35  ± 5.67	9.96 ± 5.38	—
Range	0–23	2–19
<5 y, n (%)	15 (45.7)	6 (24)
5–11 y	8 (22.8)	11 (44)
≥11 y	11 (31.5)	8 (32)
Mean last year exposure, mSv ± S.D.	69.7 ± 31.5	2.17 ± 1.9	—
Range	20–160	0.57–7.4
Mean last 5 y exposure, mSv ± S.D.	153.4 ± 28.2	11.84 ± 9.07	—
Range	50–186	1.38–35.8

Group A: industrial radiographers who had dosimetrically recorded exposure doses higher than annual dose limit of 20 mSv.

Group B: industrial radiographers who had no dosimetrically recorded exposure dose higher than annual dose limit of 20 mSv.

Table 2.

The mean values of MN, NPBs, NBUDs and NDI observed per 1000 binucleated cells in two groups of industrial radiographers and controls.

	Industrial radiographers		Control
	A	B
Mean MN frequency ± S.D. (‰)	30.32 ± 11.15a	25.48 ± 12.18b	8.40 ± 3.67c
Range	8–70	7–53	2–18
p-value	a,c0.000	a,b0.049	b,c0.000
Mean NPBs frequency ± S.D. (‰)	2.52 ± 1.92a	1 ± 0.56b	0.12 ± 0.33c
Range	0–8	0–3	0–1
p-value	a,c0.000	a,b0.000	b,c0.058
Mean NBUD frequency ± S.D. (‰)	21.29 ± 17.14a	16.41 ± 12.36b	5.80 ± 4.84c
Range	3–89	2–85	0–21
p-value	a,c0.000	a.b0.002	b,c0.004
Mean NDI frequency ± S.D. (‰)	1.7 ± 0.02a	1.81 ± 0.064b	1.83 ± 0.03c
Range	1.69–1.86	1.76–1.95	1.78–1.9
p-value	a,c0.000	a,b0.11	b,c0.3

One-way ANOVA was used for differences between groups.

^aGroup A of industrial radiographers.

^bGroup B of industrial radiographers.

^cControl.

Other cytogenetic endpoints such as NPBs and NBUD were significantly higher in the workers than in the controls and the NDI frequency in the group A is significantly lower than in the controls (Table 2).

Furthermore, between the two groups of workers, group A had significantly higher frequencies of NPBs and NBUD than in the group B (2.52 ± 1.92‰ vs 1 ± 0.56‰, p = 0.000 and 21.29 ± 17.14‰ vs 16.41 ± 12.36‰, p = 0.002, respectively), however there were no significant differences in NDI frequency between the both groups of workers (Table 2).

As shown in Table 3, there were no statistically significant differences in MN frequency in relation to age, smoking status and duration of exposure in the two groups of industrial radiographers. Even when duration of exposure divided to three groups (<5 y, 5–11 y and ≥11 y) still no significant differences were observed. However, in the control group, subjects with the age of >30 y have significantly higher MN frequencies compared with the subjects with the age of ≤30 y (9.45±3.71‰ vs 6.81±3.05‰, p = 0.02). Smoking had no effect on the MN frequency in the controls.

Table 3.

The mean MN frequency of industrial radiographers and control in relation to the smoking status, age and duration of exposure.

		Control			Radiographers
					A			B
		Mean MN	S.D.	p-value	Mean MN	S.D.	p-value	Mean MN	S.D.	p-value
Smoking status	Current	7.50	2.08	nsa	34.58	15.25	nsb	18.75	6.89	ns
	Never	8.50	3.81		28	7.54		26.76	12.66
Age	≤30 y	6.81	3.05	0.02	30.54	8.58	ns	26.88	11.80	ns
	>30 y	9.45	3.71		29.91	15.22		24.68	12.71
Duration of exposure	≤5 y	—	—	—	28.33	8.98	nsc	30.66	9.70	ns
	5–11 y	—	—		36.12	17.15		20.54	12.62
	>11 y	—	—		28.81	7.48		28.37	11.98

^ans: no significant.

^bt-test was used for differences between two groups.

^cOne-way ANOVA was used for differences between groups.

Table 4 presents the mean MN frequencies of industrial radiographers in relation to the last 1 y and last 5 y of exposure. The results did not show any significant correlation between MN frequency and annual dose in the two groups of industrial radiographers (p > 0.05). However, multiple regression showed that there was a significantly higher MN frequency in the industrial radiographers of group A who were exposed to >100 mSv doses in the last 5 y compared with individuals with ≤100 mSv (34.81 ± 12.7‰ vs 26.33 ± 7.94‰, p = 0.024). However, this effect was not found in the B group of exposed workers.

Table 4.

The mean MN frequency of industrial radiographers in relation to the last 1 y and last 5 y of exposure.

	A					B
	Dose (mSv)	Mean	S.D.	p-value	β	Dose (mSv)	Mean	S.D.	p-value	β
Last 1 y exposure	≤20	29.64	8.73	0.79	0.04	≤1.5	22.53	10.79	0.21	0.25
	>20	30.68	13.75			>1.5	28.66	13.25
Last 5 y exposure	≤100	26.33	7.94	0.024	0.38	≤5	24.27	11.64	0.67	0.08
	>100	34.81	12.7			>5	26.46	13.48

DISCUSSION

In the present study, the cytogenetic effects of occupational radiation exposure were evaluated by using cytokinesis-block MN assay in the industrial radiographers. Generally, the results show significant higher MN frequencies in the industrial radiographers than in the controls. The enhancement of MN frequency in the exposed subjects agrees with several cytogenetic investigations using the CBMN assay and dealing with radiation workers ^{(16, 23–25)}.

Our results show borderline statistically significant higher MN frequency in the group A of industrial radiographers compared with the group B, although the mean frequencies of NBUDs and NPBs in the group A were significantly higher than in the group B. However, the mean NDI value had no significantly differences in two groups. NDI could be used to define cell-cycle progression of the lymphocytes after mitogenic stimulation and could be frequently employed as a useful research tool for understanding the cell cycling kinetics of the cultures specially after radiation exposure^(6,26). In addition, NPBs and NBUDs show the spectrum of DNA damage events detectable by the CBMN assay⁽⁸⁾ and in this study illustrate more DNA damages in two groups of radiation workers than in the controls. It has been explained that NPBs may originate from dicentric chromosomes or chromatids caused by mis-repair of DNA breaks or due to telomere end fusions caused by telomere dysfunction. Also, NPBs may be made by incomplete separation of chromatids during the metaphase to anaphase transition^{(8, 22, 27)}. Studies show that NPBs are an important additional measure of radiation-induced damage⁽²⁸⁾. Studies demonstrate that NBUDs arise from nuclear removal of unresolved DNA repair complexes and excess amplified DNA; they may also be made by breakage of NPBs^{(7, 8, 22)}.

As regard to age, MN values were higher in the control group with age of >35 y in comparison with ≤35 y but such an effect was not found in the exposed subjects. This result is consistent with other large scale studies of the spontaneous incidence of MN ^{(14, 29–31)}. Furthermore, Maffei et al. have been shown MN frequencies tend to rise with age in hospital radiation workers and control, although a significant association emerge only in the control group⁽³²⁾. The increase in baseline MN frequencies as a result of the age effect may reflect a progressive enhancment in spontaneous chromosome instability associated with an accumulation of DNA damage due to an age-related decline in DNA repair capacity and can be attributed almost totally to centromere-positive MN reflecting an increased chromosome loss with age. The X-chromosome is almost completely responsible for this spontaneously occurring chromosome loss⁽¹⁷⁾.

Our findings show a significantly higher MN frequency in the industrial radiographers of group A, who were exposed to >100 mSv doses in the last 5 y compared with the individuals with ≤100 mSv. Such influence has been reported by several studies that chromosome aberrations and MN frequencies increased with the cumulative doses of ionizing radiation^{(13, 25, 33)}. However, this effect was not found in the B group of exposed workers.

In this study, occupational dosimetry records over the last 5 y ranged from 1.38 to 35.8 mSv for group B that is lower than the annual dose limits⁽¹⁹⁾. As expected, our results indicate that the frequency of MN is significantly higher in the group B than in the matched controls (p = 0.000).

In conclusion, present findings show the increased level of cytogenetic damages in the industrial radiographers compared with the controls and represent that even workers exposed to low levels of ionizing radiation, within permissible dose levels are subject to elevated frequencies of MN and other biomarkers of DNA damages such as NPBs and NBUD.

Therefore, cytogenetic biomonitoring of radiation workers is important and essential part of human health and radiation protection program beside physical dosimetry procedures for minimizing exposure to radiation at the workplace as low as possible.

Also, the high levels of DNA damages in industrial radiographers confirm the importance of the surveying radiation safety of equipment and periodic training of workers for improvement of safety and radiation protection.

ACKNOWLEDGEMENT

Special thanks are expressed to all industrial radiographers.

FUNDING

This study has been supported by Tehran University of Medical Sciences. Grant no: 27610.