Abstract
The present study investigates whether the chronic low-dose radiation exposure induces an in vivo radio-adaptive response in individuals from high-level natural radiation areas (HLNRA) of the Kerala coast. Peripheral blood samples from 54 adult male individuals aged between 26 and 65 years were collected for the study with written informed consent. Each of the whole blood sample was divided into three, one was sham irradiated, second and third was exposed to challenging doses of 1.0 and 2.0 Gy gamma radiation, respectively. Cytokinesis-block micronucleus (CBMN) assay was employed to study the radio-adaptive response. Seventeen individuals were from normal-level natural radiation area (NLNRA ≤1.5 mGy/year) and 37 from HLNRA (> 1.5 mGy/year). Based on the annual dose received, individuals from HLNRA were further classified into low-dose group (LDG, 1.51–5.0 mGy/year, N = 19) and high-dose group (HDG >5.0 mGy/year, N = 18). Basal frequency of micronucleus (MN) was comparable across the three dose groups (NLNRA, LDG and HDG, P = 0.64). Age of the individuals showed a significant effect on the frequency of MN after challenging dose exposures. The mean frequency of MN was significantly lower in elder (>40 years) individuals from HDG of HLNRA as compared to the young (≤40 years) individuals after 1.0 Gy (P < 0.001) and 2.0 Gy (P = 0.002) of challenging doses. However, young and elder individuals within NLNRA and LDG of HLNRA showed similar frequency of MN after the challenging dose exposures. Thus, increased level of chronic low-dose radiation (>5.0 mGy/year) seems to act as a priming dose resulting in the induction of an in vivo radio-adaptive response in elder individuals of the Kerala coast.
Introduction
Adaptive response is a biological phenomenon in which the cells exposed to low dose of a physical or chemical genotoxic agent (priming dose) become less susceptible to the damage induced on subsequent exposure to higher challenging dose of the same or another genotoxic agent. The phenomenon is called radio-adaptation when the primed cells are challenged with high dose of ionizing radiation and shows a reduced effect of the challenging dose (1). The radio-adaptive response in human lymphocytes was first reported by Olivieri et al. (2), where cells pretreated with tritiated thymidine showed decrease in chromosome aberration frequency on subsequent exposure to high dose X-rays. Different experimental approaches were used to study the induction of radio-adaptive response in humans that include pretreatment of human blood lymphocytes to low dose of radiation, referred to as priming dose, under in vitro or in vivo conditions, followed by a challenging treatment with ionizing radiation. In vitro studies investigated the potential of an acute priming dose in the range of 0.01–0.5 Gy (3). However, all in vitro priming exposures did not result in the induction of radio-adaptation (4,5). The induction of radio-adaptive response appeared to be influenced by the time interval between priming and challenging dose, the dose and dose rate of the ionizing radiation and, inter and intra individual variations (6–10).
The potential of chronic low-dose radiation to induce an adaptive response in humans was investigated in a limited number of studies. The studies were mainly carried out in peripheral blood lymphocytes of individuals occupationally exposed to radiation or in adults and children of areas contaminated due to Chernobyl accident (11–16). Studies on individuals occupationally exposed to ionizing radiation indicated that chronic low-dose radiation might induce adaptation in humans (11–14). Peripheral blood lymphocytes from children living in the contaminated areas due to Chernobyl accident showed no significant difference in chromosome aberration as compared to the control group when challenged with 1.5 Gy gamma rays, indicating absence of adaptive response (15). However, adaptive response was observed when human lymphocytes exposed to the fallout of Chernobyl accident were challenged with 2.5 µg/ml of bleomycin, a glycopeptide causing DNA double strand breaks that mimic the effect of ionizing radiation (16).
Studies to assess the potential of chronic low-dose radiation to induce in vivo radio-adaptive response were also carried out in individuals from high-level natural radiation areas (HLNRA) (17,18). As compared to the individuals occupationally exposed to ionizing radiation, inhabitants of HLNRA are exposed to chronic low-dose radiation for generations and during all stages of their life from conception to death. The coastal belt of Kerala state in southwest India with elevated levels of natural radiation has been inhabited for over 1000 years (19,20) and provides unique opportunity to study the potential of chronic low-dose radiation in inducing an in vivo radio-adaptation in human. The area has deposits of radioactive monazite sand in a narrow strip starting from Neendakara Panchayat in the south to Purakkad in the north (55 km long and 0.5 km wide). The higher level of natural radiation in this area is mainly due to thorium and its decay products present in the monazite sand. Due to uneven deposits of monazite sand, the ambient radiation level ranges from < 1.0 to 45.0 mGy/year averaging about 4.0 mGy/year (21,22).
Several studies have been conducted in this population to understand the biological and health effects of high level natural radiation exposures. Cytogenetic analysis of plants from the monazite bearing areas revealed meiotic abnormalities associated with external radiation level (23). Morphological measurements of bones and teeth of wild rats had not shown any genetic effects attributable to high level of natural radiation (24). A demographic survey based on a population of 70 000 individuals did not show any association of infertility or infant mortality rate with high background radiation (25). Incidence of congenital malformations, chromosome anomalies among consecutive newborns and cancer in individuals of the area did not reveal any significant difference between HLNRA and normal-level natural radiation areas (NLNRA) (26–28). A case–control study on cleft lip or palate and mental retardation (29), studies on the frequency of micronuclei in newborns and adults (30,31), telomere length attrition in adults and newborns (32,33) and spontaneous DNA strand breaks in PBMCs from healthy adults (34) also did not indicate significant differences between HLNRA and NLNRA individuals. Lack of increased level of DNA double strand breaks was observed in HLNRA population as compared to NLNRA and low-dose group of HLNRA (35). A study on DNA damage and repair kinetics of DNA strand breaks using alkaline comet assay suggested that in vivo chronic low-level radiation may induce an adaptive response (36).
Various biomarkers such as cell lethality, apoptosis, chromosome aberration, micronucleus (MN), mutation induction, malignant transformation, DNA repair, level of gene and protein expression have been used to study the phenomenon of radio-adaptive response in humans (9,10,37–43). The cytokinesis-block micronucleus (CBMN) assay is a sensitive, fast and reliable technique to quantify genetic damage in individuals (44). The technique was employed in several studies as an alternative approach for quantifying chromosome damage, as it offers a measure of both chromosome breakage and loss (44–46). The present study investigates the role of chronic low-dose radiation on the induction of an in vivo radio-adaptive response in adult male individuals from HLNRA of Kerala, using CBMN assay.
Materials and methods
Study group and blood sample collection
The study was carried out in 54 adult male individuals with an average age of 41.2 ± 8.8 years (mean ± SD), which ranged from 26 to 65 years. Of this, 17 individuals belonged to NLNRA (≤1.5 mGy/year) with an average age of 40.6 ± 9.5 (range, 28–55 years) and 37 individuals belonged to HLNRA (>1.5 mGy/year) with an average age of 41.5 ± 8.6 (range, 26–65 years). Individuals from HLNRA were further classified into two groups based on annual dose received: low-dose group (LDG, 1.51–5.0 mGy/year) and high-dose group (HDG, >5.0 mGy/year) with mean annual doses of 2.66 mGy/year (N = 19, range, 1.53–4.83 mGy/year) and 12.64 mGy/year (N =18, range, 7.21–22.71 mGy/year), respectively.
About 5 ml of whole blood was collected from each individual by venipuncture using heparin-coated vacutainer tubes (BD Vacutainer). Samples were collected after obtaining written informed consent of all the donors participating in this study. The procedure was approved by the medical ethic committee, Bhabha Atomic Research Centre (BARC), Trombay, Mumbai. Immediately after collection, the blood samples were transported to the laboratory in refrigerated conditions and were processed.
Dosimetry
The gamma radiation level in each donor’s house was measured using a halogen quenched Geiger Muller (GM) tube-based survey meter consisting of a GM tube and a microprocessor-based digital display (Type ER-709, Nucleonix Systems, India). Measurements were done at a height of 1 m inside (the main room having maximum occupancy) and outside (near the entrance) of each house on the same day of blood sample collection between 10.30 a.m. and 11.30 a.m. The mean of three readings was taken for each measurement. The radiation exposure in air (µR/h) due to γ-rays was converted to annual dose (mGy/year), using a conversion factor of 0.0765 [= 0.873 × 24 h × 365 days ×10–5, where 0.873 is the conversion factor for exposure to absorbed dose in free air (air kerma)]. The individual dose was derived as sum of 0.5 × the annual indoor dose and 0.5 × the annual outdoor dose. The coefficient of 0.5 is the occupancy factor for both indoor and outdoor for male subjects between 25 and 50 years of age, as estimated by Nair et al. (47).
Irradiation
Whole blood samples from each individual were divided into three aliquots and marked as I, II and III. Aliquot I was kept as control and was sham irradiated. Aliquots II and III was exposed to 1.0 and 2.0 Gy gamma radiation, respectively, using a Cobalt-60 gamma source at a dose rate of 0.8 Gy/min (Low-dose irradiator-2000, BRIT, India).
Cytokinesis-block micronucleus assay
Whole blood cultures were set up by adding the blood sample (0.5 ml) to RPMI 1640 (4.5 ml), supplemented with benzyl penicillin (100 U/ml), streptomycin (100 µg/ml), 1% l-glutamine (200 mM), 10% FCS and PHA (10 µg/ml) followed by incubation at 37°C. Cytochalasin B in dimethyl sulfoxide (6 µg/ml) was added to the culture at 44 h and processed at 72 h with mild hypotonic treatment using potassium chloride (0.075 M), followed by fixing the cells in 3:1 methanol and acetic acid. The cell suspension was dropped carefully on to pre-cleaned, ice-chilled glass slides and allowed to dry at room temperature. The slides prepared from each culture were coded, stained with 2% Giemsa at pH 6.8 and mounted using DPX. The slides were scored as per the standard criteria under bright-field microscope (Olympus BX-60) at 400× magnification for binucleated (BN) cells with well preserved cytoplasm. Four experienced cytogeneticists were involved in scoring MN following standard criteria (48,49). Doubtful BN cells with MN were verified and confirmed by all the cytogeneticists at 1000× magnification. An average of 1000 BN cells were analysed for each dose point. Frequency of MN was calculated per 1000 BN cells. Basal frequency of MN was estimated in all the individuals studied.
Nuclear division index
In order to understand whether the in vitro gamma irradiation affect the cell cycle, the nuclear division index (NDI) was estimated from a random subset of 32 individuals [NLNRA, N = 9; HLNRA, N = 23 (LDG, N = 14; HDG, N = 9)] using the formula NDI = (M1 + 2M2 + 3M3 + 4M4)/N; where M1–M4 indicate the number of cells with one to four nuclei, respectively, and N is the total number of cells scored (50). NDI was estimated from a total of 500 cells at each dose point.
Statistical methods
The basal frequency of MN across the three dose groups was compared using ANOVA. The possibility of chronic low-dose radiation acting as an in vivo priming dose was assessed by comparing MN yield after 1.0 and 2.0 Gy irradiation in blood samples of individuals from NLNRA, LDG and HDG from HLNRA, after adjusting for the effect of age using ANCOVA. The frequency of MN between young and elder individuals classified using the cut-off point of 40 years, the mean age among the individuals studied, was compared using student’s t-test within each dose group. Grouping of individuals using 40 years cut-off was followed by Nefic and Handzic (51) in their study on micronuclei. The P values less than 0.05 were considered as significant. Multivariate analysis of variance was employed to compare NDI at 0, 1.0 and 2.0 Gy across all the three dose groups of NLNRA; LDG and HDG of HLNRA and between the age groups of ≤40 and >40. Statistical analysis was carried out using STATISTICA software, version 9.1 (52).
Results
The basal and radiation induced (1.0 and 2.0 Gy challenging dose) frequency of MN obtained in individuals from NLNRA and, LDG and HDG of HLNRA is given in Table 1. Mean basal frequency of MN in individuals from NLNRA, LDG and HDG was 11.45 ± 1.53, 13.77 ± 1.91 and 12.31 ± 1.74 per 1000 BN cells, respectively. ANOVA showed that the basal frequency of MN was comparable across the three dose groups (P = 0.64).
Frequency of micronuclei/1000 BN cells at 0, 1.0 and 2.0 Gy of challenging dose
| Area | N | Age (mean ± SD) | Dose (mGy/year) | Mean number of MN per 1000 BN cells ± SE | ||
|---|---|---|---|---|---|---|
| Basal (0 Gy) | Challenging dose | |||||
| 1.0 Gy | 2.0 Gy | |||||
| NLNRA (≤1.50 mGy/year) | 17 | 40.6 ± 9.5 (28–55) | 1.31 ± 0.08 (1.19–1.46) | 11.45 ± 1.53 | 238.75 ± 20.26 | 616.35 ± 42.71 |
| HLNRA (LDG) (1.51–5.0 mGy/year) | 19 | 42.1 ± 7.1 (32–55) | 2.66 ± 1.01 (1.53–4.83) | 13.77 ± 1.91 | 235.50 ± 15.89 | 569.98 ± 39.43 |
| HLNRA (HDG) (>5.0 mGy/year) | 18 | 40.8 ± 10.1 (26–65) | 12.64 ± 3.99 (7.21–22.71) | 12.31 ± 1.74 | 226.99 ± 21.1 | 631.02 ± 42.35 |
| HLNRA (LDG + HDG) | 37 | 41.5 ± 8.6 (26–65) | 7.51 ± 5.79 (1.53–22.71) | 13.06 ± 1.28 | 231.36 ± 12.95 | 559.67 ± 28.93 |
| Area | N | Age (mean ± SD) | Dose (mGy/year) | Mean number of MN per 1000 BN cells ± SE | ||
|---|---|---|---|---|---|---|
| Basal (0 Gy) | Challenging dose | |||||
| 1.0 Gy | 2.0 Gy | |||||
| NLNRA (≤1.50 mGy/year) | 17 | 40.6 ± 9.5 (28–55) | 1.31 ± 0.08 (1.19–1.46) | 11.45 ± 1.53 | 238.75 ± 20.26 | 616.35 ± 42.71 |
| HLNRA (LDG) (1.51–5.0 mGy/year) | 19 | 42.1 ± 7.1 (32–55) | 2.66 ± 1.01 (1.53–4.83) | 13.77 ± 1.91 | 235.50 ± 15.89 | 569.98 ± 39.43 |
| HLNRA (HDG) (>5.0 mGy/year) | 18 | 40.8 ± 10.1 (26–65) | 12.64 ± 3.99 (7.21–22.71) | 12.31 ± 1.74 | 226.99 ± 21.1 | 631.02 ± 42.35 |
| HLNRA (LDG + HDG) | 37 | 41.5 ± 8.6 (26–65) | 7.51 ± 5.79 (1.53–22.71) | 13.06 ± 1.28 | 231.36 ± 12.95 | 559.67 ± 28.93 |
Values in parenthesis indicate range. LDG, low-dose group; HDG, high-dose group; BN, binucleated; HLNRA, high-level natural radiation area; MN, micronuclei; NLNRA, normal level natural radiation area.
Frequency of micronuclei/1000 BN cells at 0, 1.0 and 2.0 Gy of challenging dose
| Area | N | Age (mean ± SD) | Dose (mGy/year) | Mean number of MN per 1000 BN cells ± SE | ||
|---|---|---|---|---|---|---|
| Basal (0 Gy) | Challenging dose | |||||
| 1.0 Gy | 2.0 Gy | |||||
| NLNRA (≤1.50 mGy/year) | 17 | 40.6 ± 9.5 (28–55) | 1.31 ± 0.08 (1.19–1.46) | 11.45 ± 1.53 | 238.75 ± 20.26 | 616.35 ± 42.71 |
| HLNRA (LDG) (1.51–5.0 mGy/year) | 19 | 42.1 ± 7.1 (32–55) | 2.66 ± 1.01 (1.53–4.83) | 13.77 ± 1.91 | 235.50 ± 15.89 | 569.98 ± 39.43 |
| HLNRA (HDG) (>5.0 mGy/year) | 18 | 40.8 ± 10.1 (26–65) | 12.64 ± 3.99 (7.21–22.71) | 12.31 ± 1.74 | 226.99 ± 21.1 | 631.02 ± 42.35 |
| HLNRA (LDG + HDG) | 37 | 41.5 ± 8.6 (26–65) | 7.51 ± 5.79 (1.53–22.71) | 13.06 ± 1.28 | 231.36 ± 12.95 | 559.67 ± 28.93 |
| Area | N | Age (mean ± SD) | Dose (mGy/year) | Mean number of MN per 1000 BN cells ± SE | ||
|---|---|---|---|---|---|---|
| Basal (0 Gy) | Challenging dose | |||||
| 1.0 Gy | 2.0 Gy | |||||
| NLNRA (≤1.50 mGy/year) | 17 | 40.6 ± 9.5 (28–55) | 1.31 ± 0.08 (1.19–1.46) | 11.45 ± 1.53 | 238.75 ± 20.26 | 616.35 ± 42.71 |
| HLNRA (LDG) (1.51–5.0 mGy/year) | 19 | 42.1 ± 7.1 (32–55) | 2.66 ± 1.01 (1.53–4.83) | 13.77 ± 1.91 | 235.50 ± 15.89 | 569.98 ± 39.43 |
| HLNRA (HDG) (>5.0 mGy/year) | 18 | 40.8 ± 10.1 (26–65) | 12.64 ± 3.99 (7.21–22.71) | 12.31 ± 1.74 | 226.99 ± 21.1 | 631.02 ± 42.35 |
| HLNRA (LDG + HDG) | 37 | 41.5 ± 8.6 (26–65) | 7.51 ± 5.79 (1.53–22.71) | 13.06 ± 1.28 | 231.36 ± 12.95 | 559.67 ± 28.93 |
Values in parenthesis indicate range. LDG, low-dose group; HDG, high-dose group; BN, binucleated; HLNRA, high-level natural radiation area; MN, micronuclei; NLNRA, normal level natural radiation area.
With the challenging dose of 1.0 Gy, the frequency of MN was 238.75 ± 20.26, 235.50 ± 15.89 and 226.99 ± 21.1 per 1000 BN cells in NLNRA, LDG and HDG individuals, respectively. The frequency of MN at a challenging dose of 2.0 Gy was found to be 616.35 ± 42.71, 569.98 ± 39.43 and 631.02 ± 42.35 per 1000 BN cells in NLNRA, LDG and HDG individuals. As shown in Figure 1, the frequency of MN induced by the challenging dose exposure was influenced by the age of the individuals. After 1.0 Gy of irradiation, there was a significant (P = 0.01) negative correlation with respect to age (y = 359.06 − 3.044 × Age) as shown in Figure 1A. A negative correlation between age and the frequency of MN (y = 814 − 5.09 × Age) was also observed after 2.0 Gy of irradiation (P = 0.06; Figure 1B). ANCOVA results shown in Table 2 indicated that the frequency of MN was statistically comparable across the three dose groups after 1.0 Gy (P = 0.87) and 2.0 Gy (P = 0.61) challenging dose exposures.
Regression analysis showing the relationship between frequency of MN and age of the individuals after 1.0 Gy (A) and 2.0 Gy (B) challenging dose exposure.
Regression analysis showing the relationship between frequency of MN and age of the individuals after 1.0 Gy (A) and 2.0 Gy (B) challenging dose exposure.
ANCOVA test on the mean frequency of MN by taking age of the individuals as continuous variable
| Source of variation | Sum of squares | d.f. | Mean Square | F | P-value |
|---|---|---|---|---|---|
| 1.0 Gy challenging dose exposure | |||||
| Dose groups (NLNRA, LDG, HDG) | 1720.3 | 2 | 860.1 | 0.145 | 0.870 |
| Age | 38542.0 | 1 | 38542.0 | 6.517 | 0.014 |
| 2.0 Gy challenging dose exposure | |||||
| Dose groups (NLNRA, LDG, HDG) | 29122.9 | 2 | 14561.5 | 0.493 | 0.610 |
| Age | 98404.0 | 1 | 98404.0 | 3.329 | 0.070 |
| Source of variation | Sum of squares | d.f. | Mean Square | F | P-value |
|---|---|---|---|---|---|
| 1.0 Gy challenging dose exposure | |||||
| Dose groups (NLNRA, LDG, HDG) | 1720.3 | 2 | 860.1 | 0.145 | 0.870 |
| Age | 38542.0 | 1 | 38542.0 | 6.517 | 0.014 |
| 2.0 Gy challenging dose exposure | |||||
| Dose groups (NLNRA, LDG, HDG) | 29122.9 | 2 | 14561.5 | 0.493 | 0.610 |
| Age | 98404.0 | 1 | 98404.0 | 3.329 | 0.070 |
d.f., degrees of freedom; HDG, high-dose group (>5.0 mGy/year); LDG, low-dose group (1.51–5.0 mGy/year); NLNRA, normal level natural radiation area (≤1.5 mGy/year).
ANCOVA test on the mean frequency of MN by taking age of the individuals as continuous variable
| Source of variation | Sum of squares | d.f. | Mean Square | F | P-value |
|---|---|---|---|---|---|
| 1.0 Gy challenging dose exposure | |||||
| Dose groups (NLNRA, LDG, HDG) | 1720.3 | 2 | 860.1 | 0.145 | 0.870 |
| Age | 38542.0 | 1 | 38542.0 | 6.517 | 0.014 |
| 2.0 Gy challenging dose exposure | |||||
| Dose groups (NLNRA, LDG, HDG) | 29122.9 | 2 | 14561.5 | 0.493 | 0.610 |
| Age | 98404.0 | 1 | 98404.0 | 3.329 | 0.070 |
| Source of variation | Sum of squares | d.f. | Mean Square | F | P-value |
|---|---|---|---|---|---|
| 1.0 Gy challenging dose exposure | |||||
| Dose groups (NLNRA, LDG, HDG) | 1720.3 | 2 | 860.1 | 0.145 | 0.870 |
| Age | 38542.0 | 1 | 38542.0 | 6.517 | 0.014 |
| 2.0 Gy challenging dose exposure | |||||
| Dose groups (NLNRA, LDG, HDG) | 29122.9 | 2 | 14561.5 | 0.493 | 0.610 |
| Age | 98404.0 | 1 | 98404.0 | 3.329 | 0.070 |
d.f., degrees of freedom; HDG, high-dose group (>5.0 mGy/year); LDG, low-dose group (1.51–5.0 mGy/year); NLNRA, normal level natural radiation area (≤1.5 mGy/year).
We have observed an influence of age on the frequency of MN after challenging dose exposures and individuals from NLNRA, LDG and HDG were further classified into young (≤ 40 years) and elder (>40 years) age groups. The mean frequency of MN in young and elder individuals after 1.0 and 2.0 Gy challenging doses in blood samples of individuals from NLNRA, LDG and HDG are given in Table 3. In each dose group, the mean frequency of MN was compared between young and elder age groups using the student’s t-test. Results indicated that the mean frequency of MN was significantly lower (P < 0.001) in elder individuals (161.28 ± 21.60) from HDG of HLNRA as compared to the young individuals (292.70 ± 18.59) after 1.0 Gy of challenging dose exposure. However, in other two dose groups (NLNRA and LDG of HLNRA), the mean frequency of MN was statistically comparable between young and elder individuals [NLNRA, P = 0.51; LDG (HLNRA), P = 0.92]. Similarly, after 2.0 Gy of challenging dose, the mean frequency of MN in elder individuals (512.26 ± 44.74) from HDG of HLNRA showed a statistically significant (P = 0.002) reduction as compared to the mean frequency of MN in young individuals (749.78 ± 45.75). The mean frequency of MN was comparable between young and elder individuals [NLNRA, P = 0.79; LDG (HLNRA), P = 0.49] in the other two dose groups. ANOVA on the frequency of MN among young individuals across the three dose groups suggested that the apparent increase in the frequency of MN among young individuals of high dose group exposed to 1.0 and 2.0 Gy compared to other two lower dose groups was not statistically significant, 1.0 Gy: F2,23 = 1.24, P = 0.309 and 2.0 Gy: F2,23 = 1.93 and P = 0.168. Also, MN frequency among the young individuals of HDG was contrasted against the average frequency of MN in NLNRA and LDG and the difference was not statistically significant, 1.0 Gy: t23 = 1.54, P = 0.139 and 2.0 Gy: t23 = 1.95, P = 0.064.
Frequency of micronuclei/1000 BN cells in individuals with ≤40 years and >40 years of age from NLNRA, LDG and HDG at 0, 1.0 and 2.0 Gy of challenging dose
| Groups | N | Age (mean ± SD) | Dose (mGy/year) | Mean number of MN per 1000 BN cells ± SE | ||
|---|---|---|---|---|---|---|
| Basal (0 Gy) | Challenging dose | |||||
| 1.0 Gy | 2.0 Gy | |||||
| NLNRA (≤1.5 mGy/year) | ||||||
| ≤40 years | 9 | 33.0 ± 4.3 | 1.34 ± 0.09 | 9.72 ± 1.69 | 252.0 ± 31.70 | 627.78 ± 64.50 |
| >40 years | 8 | 49.1 ± 5.3 | 1.28 ± 0.07 | 13.39 ± 2.57 | 223.83 ± 25.34 | 603.50 ± 58.86 |
| HLNRA (LDG, 1.51–5.0 mGy/year) | ||||||
| ≤40 years | 8 | 35.1 ± 2.8 | 2.90 ± 1.03 | 13.69 ± 3.87 | 237.48 ± 24.93 | 603.12 ± 58.48 |
| >40 years | 11 | 47.2 ± 4.2 | 2.49 ± 1.01 | 13.84 ± 1.93 | 234.07 ± 21.64 | 545.87 ± 54.36 |
| HLNRA (HDG, >5.0 mGy/year) | ||||||
| ≤40 years | 9 | 32.9 ± 5.2 | 13.21 ± 3.73 | 14.11 ± 2.97 | 292.70 ± 18.59 | 749.78 ± 45.75 |
| >40 years | 9 | 48.7 ± 7.1 | 12.06 ± 4.36 | 10.50 ± 1.81 | 161.28 ± 21.60† | 512.26 ± 44.74‡ |
| Groups | N | Age (mean ± SD) | Dose (mGy/year) | Mean number of MN per 1000 BN cells ± SE | ||
|---|---|---|---|---|---|---|
| Basal (0 Gy) | Challenging dose | |||||
| 1.0 Gy | 2.0 Gy | |||||
| NLNRA (≤1.5 mGy/year) | ||||||
| ≤40 years | 9 | 33.0 ± 4.3 | 1.34 ± 0.09 | 9.72 ± 1.69 | 252.0 ± 31.70 | 627.78 ± 64.50 |
| >40 years | 8 | 49.1 ± 5.3 | 1.28 ± 0.07 | 13.39 ± 2.57 | 223.83 ± 25.34 | 603.50 ± 58.86 |
| HLNRA (LDG, 1.51–5.0 mGy/year) | ||||||
| ≤40 years | 8 | 35.1 ± 2.8 | 2.90 ± 1.03 | 13.69 ± 3.87 | 237.48 ± 24.93 | 603.12 ± 58.48 |
| >40 years | 11 | 47.2 ± 4.2 | 2.49 ± 1.01 | 13.84 ± 1.93 | 234.07 ± 21.64 | 545.87 ± 54.36 |
| HLNRA (HDG, >5.0 mGy/year) | ||||||
| ≤40 years | 9 | 32.9 ± 5.2 | 13.21 ± 3.73 | 14.11 ± 2.97 | 292.70 ± 18.59 | 749.78 ± 45.75 |
| >40 years | 9 | 48.7 ± 7.1 | 12.06 ± 4.36 | 10.50 ± 1.81 | 161.28 ± 21.60† | 512.26 ± 44.74‡ |
BN, binucleated; HDG, high-dose group; LDG, low-dose group; MN, micronuclei; NLNRA, normal level natural radiation area.
†P < 0.001; ‡P = 0.002, when compared with individuals having ≤40 years of age.
Frequency of micronuclei/1000 BN cells in individuals with ≤40 years and >40 years of age from NLNRA, LDG and HDG at 0, 1.0 and 2.0 Gy of challenging dose
| Groups | N | Age (mean ± SD) | Dose (mGy/year) | Mean number of MN per 1000 BN cells ± SE | ||
|---|---|---|---|---|---|---|
| Basal (0 Gy) | Challenging dose | |||||
| 1.0 Gy | 2.0 Gy | |||||
| NLNRA (≤1.5 mGy/year) | ||||||
| ≤40 years | 9 | 33.0 ± 4.3 | 1.34 ± 0.09 | 9.72 ± 1.69 | 252.0 ± 31.70 | 627.78 ± 64.50 |
| >40 years | 8 | 49.1 ± 5.3 | 1.28 ± 0.07 | 13.39 ± 2.57 | 223.83 ± 25.34 | 603.50 ± 58.86 |
| HLNRA (LDG, 1.51–5.0 mGy/year) | ||||||
| ≤40 years | 8 | 35.1 ± 2.8 | 2.90 ± 1.03 | 13.69 ± 3.87 | 237.48 ± 24.93 | 603.12 ± 58.48 |
| >40 years | 11 | 47.2 ± 4.2 | 2.49 ± 1.01 | 13.84 ± 1.93 | 234.07 ± 21.64 | 545.87 ± 54.36 |
| HLNRA (HDG, >5.0 mGy/year) | ||||||
| ≤40 years | 9 | 32.9 ± 5.2 | 13.21 ± 3.73 | 14.11 ± 2.97 | 292.70 ± 18.59 | 749.78 ± 45.75 |
| >40 years | 9 | 48.7 ± 7.1 | 12.06 ± 4.36 | 10.50 ± 1.81 | 161.28 ± 21.60† | 512.26 ± 44.74‡ |
| Groups | N | Age (mean ± SD) | Dose (mGy/year) | Mean number of MN per 1000 BN cells ± SE | ||
|---|---|---|---|---|---|---|
| Basal (0 Gy) | Challenging dose | |||||
| 1.0 Gy | 2.0 Gy | |||||
| NLNRA (≤1.5 mGy/year) | ||||||
| ≤40 years | 9 | 33.0 ± 4.3 | 1.34 ± 0.09 | 9.72 ± 1.69 | 252.0 ± 31.70 | 627.78 ± 64.50 |
| >40 years | 8 | 49.1 ± 5.3 | 1.28 ± 0.07 | 13.39 ± 2.57 | 223.83 ± 25.34 | 603.50 ± 58.86 |
| HLNRA (LDG, 1.51–5.0 mGy/year) | ||||||
| ≤40 years | 8 | 35.1 ± 2.8 | 2.90 ± 1.03 | 13.69 ± 3.87 | 237.48 ± 24.93 | 603.12 ± 58.48 |
| >40 years | 11 | 47.2 ± 4.2 | 2.49 ± 1.01 | 13.84 ± 1.93 | 234.07 ± 21.64 | 545.87 ± 54.36 |
| HLNRA (HDG, >5.0 mGy/year) | ||||||
| ≤40 years | 9 | 32.9 ± 5.2 | 13.21 ± 3.73 | 14.11 ± 2.97 | 292.70 ± 18.59 | 749.78 ± 45.75 |
| >40 years | 9 | 48.7 ± 7.1 | 12.06 ± 4.36 | 10.50 ± 1.81 | 161.28 ± 21.60† | 512.26 ± 44.74‡ |
BN, binucleated; HDG, high-dose group; LDG, low-dose group; MN, micronuclei; NLNRA, normal level natural radiation area.
†P < 0.001; ‡P = 0.002, when compared with individuals having ≤40 years of age.
NDI showed decreasing trend with increasing dose of gamma radiation. The mean (SD) NDI among samples irradiated with 0, 1.0 and 2.0 Gy of gamma radiation was 1.34 (0.16), 1.27 (0.13) and 1.17 (0.09), respectively. Multivariate analysis on NDI at 0, 1.0 and 2.0 Gy did not suggest any difference across the three dose groups of NLNRA, LDG and HDG of HLNRA (Wilks Lambda = 0.832, P = 0.526) or between age groups ≤40 years and >40 years (Wilks Lambda = 0.863, P = 0.239).
Discussion
The objective of the present study was to evaluate whether the chronic low-dose radiation prevailing in the Kerala coast provides an in vivo priming exposure that allows an adaptation to a subsequent higher radiation exposure in adult male individuals residing in the area. The CBMN assay was employed to compare the basal and gamma radiation (either 1.0 or 2.0 Gy) induced frequency of MN in peripheral blood mononuclear cells (PBMCs) of individuals from NLNRA and, low and high dose groups of HLNRA. Several studies have investigated the genotoxic effect of low-dose radiation in individuals by estimating the frequency of MN in PBMCs (53–57). However, studies focusing on the priming effect of in vivo chronic low-dose radiation on radio-adaptive response are limited (11–13,18,36). In the present study, challenging doses of 1.0 or 2.0 Gy were given to the blood samples immediately after collection and hence majority of the blood lymphocytes would be receiving the dose essentially in the G0 phase of the cell cycle. Reports on the induction of radio-adaptive response in G0 lymphocytes are equivocal. While some of the studies failed to observe radio-adaptive response in quiescent G0 lymphocytes (39,58,59), others reported induction of radio-adaptive response (9,10,17,18,36,60).
We have observed statistically comparable basal frequency of MN in the PBMCs of individuals from NLNRA and, LDG and HDG from HLNRA. This might indicate that the high level natural radiation of Kerala coast did not influence the basal frequency of MN. A recent report from our laboratory indicated similar frequency of MN in adult individuals from NLNRA and HLNRA (31). Similarly, earlier studies on newborns from this area reported statistically comparable frequencies of MN and chromosome aberrations including dicentrics in NLNRA and HLNRA individuals (27,30). A study on 27 individuals (13 from high and 14 from normal background radiation areas) from Ramsar, Iran, showed statistically comparable basal frequency of MN between control and exposed groups (18). No statistically significant effect of occupational radiation exposure on the frequency of MN was found in nuclear radiation workers (13,53). Nevertheless, some of the cytogenetic studies performed in the high background radiation areas of Ramsar have shown increased frequencies of unstable chromosome aberrations (61,62). In addition, higher frequency of MN were reported in medical radiation workers and in interventional cardiologists compared to non exposed individuals (11,54,63).
A significant positive correlation between age and the basal frequency of MN was reported in several studies (53,64,65). Interestingly, we observed a negative correlation between age and the frequency of MN after challenging dose exposures. Individuals were classified in to young and elder individuals using the cut-off point of 40 years, the mean age of individuals in the age group of 26–65 years. In the stratified analysis, we found statistically significant lower frequency of MN in elder individuals from high dose group (HDG >5.0 mGy/year) of HLNRA on both 1.0 and 2.0 Gy challenging dose exposure as compared to the young individuals. Several studies have reported that a priming dose may result in a reduction in the frequency of MN after the challenging dose (18,66,67). In agreement with these reports, a reduction in the frequency of MN was observed in the elder individuals of HDG from HLNRA after the challenging treatment, indicating the possibility of chronic low-dose radiation acting as a priming dose and inducing an in vivo radio-adaptive response. Lower initial levels of DNA strand breaks after 2.0 or 4.0 Gy of challenging dose of gamma radiation in individuals of HDG from HLNRA, indicating an in vivo radio-adaptive response, was reported previously (36). Induction of radio adaptive response due to chronic ionizing radiation exposure after environmental, occupational, and nuclear accidents in humans has been reviewed (3,68,69). However, there was no significant difference in the frequency of MN between young and elder individuals within NLNRA and within LDG of HLNRA after challenging dose exposure. Although young individuals of HDG from HLNRA showed an apparent increase in the frequency of MN compared to the young individuals from LDG and NLNRA after 1.0 or 2.0 Gy of gamma irradiation, the difference was not statistically significant.
It has been shown that ageing leads to either non-induction or decrease of adaptive response in humans (70). One of the hypotheses suggested that the antioxidant defense mechanism that is already induced in aged cells by environmental and chronic oxidative stress may not recognize low-dose irradiation as a redox imbalance. However, extensive studies on the activities of antioxidant enzymes such as catalase, glutathione peroxidase and glutathione reductase in young and aged rat glial cells after in vitro pre-irradiation and subsequent challenging dose exposure could not prove the hypothesis (71). On the other hand, our study suggested a probable induction of radio-adaptive response in elder individuals exposed to chronic low-dose radiation of >5.0 mGy/year as compared to the elder individuals from LDG and NLNRA. The radio-adaptive response observed among elder individuals may indicate that the quantity of initial lesions required to evoke adaptation effectively is an important parameter. Thus, it might be plausible that the dose and dose rate of the priming dose influence the induction of cellular and /or molecular responses upon a subsequent challenging dose (72).
The possibility of in vitro gamma irradiation affecting the cell cycle was evaluated by estimating the NDI. We found that the NDI decreases with increasing dose of gamma radiation, but the same may not affect relationship between MN yield, age of the individuals and background dose level as the decrease in NDI was similar in individuals from all the three dose groups (NLNRA, LDG and HDG) and also in young and elder individuals.
In conclusion, preliminary results obtained from our study suggest induction of an in vivo radio-adaptive response in elder male individuals from HLNRA who received an annual chronic dose of >5.0 mGy/year. Absence of such an observation in male individuals from LDG of HLNRA is intriguing and need to be probed further to discern the individual and interactive relationship of age and background radiation exposure on the induction of micronuclei. However, it would be interesting to conduct radio-adaptive response among female individuals from this area. Studies using other biological end-points such as mRNA, miRNA and protein expression profile may be explored to understand the radio-adaptation in individuals from HLNRA.
Acknowledgements
The authors are grateful to all the volunteers who donated blood samples for this study. Authors thank the efforts of M. P. George, who motivated the individuals from the normal and high-level natural radiation areas to participate in this study. Cooperation of the laboratory staff of Government hospitals, Kollam, Kerala, for blood sample collection is highly appreciated. Technical help of P.S. Mohan, R. Prabhakaran, K. Saseendran and logistic support of all the other staff members of our laboratory is acknowledged.
Conflict of interest statement: None declared.
