Radio-adaptive response in peripheral blood lymphocytes of individuals residing in high-level natural radiation areas of Kerala in the southwest coast of India.

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.

Sex ratio at birth: scenario from normal- and high-level natural radiation areas of Kerala coast in south-west India.

Newborns were monitored for congenital malformations in four government hospitals located in high-level (ambient dose >1.5 mGy/year) and normal-level (≤ 1.5 mGy/year) natural radiation areas of Kerala, India, from August 1995 to December 2012. Sex ratio at birth (SRB) among live singleton newborns and among previous children, if any, of their mothers without history of any abortion, stillbirth or twins is reported here. In the absence of environmental stress or selective abortion of females, global average of SRB is about 1050 males to 1000 females. A total of 151,478 singleton, 1031 twins, 12 triplets and 1 quadruplet deliveries were monitored during the study period. Sex ratio among live singleton newborns was 1046 males (95 % CI 1036-1057) for 1000 females (77,153 males:73,730 females) and was comparable to the global average. It was similar in high-level and normal-level radiation areas of Kerala with SRB of 1050 and 1041, respectively. It was consistently more than 1000 and had no association with background radiation levels, maternal and paternal age at birth, parental age difference, gravida status, ethnicity, consanguinity or year of birth. Analysis of SRB of the children of 139,556 women whose reproductive histories were available suggested that couples having male child were likely to opt for more children and this, together with enhanced rate of males at all birth order, was skewing the overall SRB in favour of male children. Though preference for male child was apparent, extreme steps of sex-selective abortion or infanticide were not prevalent.

Effects of chronic low level radiation in the population residing in the high level natural radiation area in Kerala, India: employing heritable DNA mutation studies.

To study the effect of chronic low level radiation, 4040 meiosis were screened at eight microsatellite and five minisatellite (2485 and 1555 meiosis respectively) marker loci in people residing in high and normal level natural radiation areas of Kerala. Variants in the repeat length of allele were considered as mutants. Mutation rates (expressed as the number of mutations observed in the total number of meiosis) were 6.4×10(-3) (16/2485) and 2.6×10(-3) (4/1555) at microsatellite and minisatellite respectively. The germline microsatellite mutation frequency of father was 1.78 times higher at 7.52×10(-3) (8/1064) compared to 4.22×10(-3) (6/1421) of mother (P=0.292, Fisher's Exact two-sided test). The paternal and maternal mutation rates at minisatellite loci were more or less similar at 2.78×10(-3) (2/719) and 2.39×10(-3) (2/836), respectively (P=1.0, Fisher's Exact two-sided test). Higher but statistically non-significant microsatellite mutation frequency was observed in HLNRA compared to NLNRA (7.25×10(-3) vs 3.64×10(-3); P=0.547). The apparent increase in the mutation rate of microsatellite loci with the increase in radiation dose was also not statistically significant. All the four minisatellite mutation observed were from HLNRA (1198 meiosis) and no mutation was observed among 357 meiosis screened from NLNRA families. All the markers used in the present study were in the non-coding region and hence mutations in these regions may not cause adverse health effects, but the study is important in understanding the effect of chronic low level radiation.

Non-linear dose response of DNA double strand breaks in response to chronic low dose radiation in individuals from high level natural radiation areas of Kerala coast.

BACKGROUND: The human population living in high level natural radiation areas (HLNRAs) of Kerala coast provide unique opportunities to study the biological effects of low dose and low dose rate ionizing radiation below 100 mGy. The level of radiation in this area varies from < 1.0 to 45 mGy/year. The areas with ≤ 1.50 mGy/year are considered as normal level natural radiation areas (NLNRA) and > 1.50 mGy/year, as high level natural radiation areas (HLNRA). The present study evaluated dose response relationship between DNA double strand breaks (DSBs) and background radiation dose in individuals residing in Kerala coast. Venous blood samples were collected from 200 individuals belonging to NLNRA (n = 50) and four dose groups of HLNRA; 1.51-5.0 mGy/year (n = 50), 5.01-10.0 mGy/year (n = 30), 10.01-15.0 mGy/year (n = 33), > 15.0 mGy/year (n = 37) with written informed consent. The mean dose of NLNRA and four HLNRA dose groups studied are 1.21 ± 0.21 (range: 0.57-1.49), 3.02 ± 0.95 (range: 1.57-4.93), 7.43 ± 1.48 (range: 5.01-9.75), 12.22 ± 1.47 (range: 10.21-14.99), 21.64 ± 6.28 (range: 15.26-39.88) mGy/year, respectively. DNA DSBs were quantified using γH2AX as a marker, where foci were counted per cell using fluorescence microscopy. RESULTS: Our results revealed that the frequency of γH2AX foci per cell was 0.090 ± 0.051 and 0.096 ± 0.051, respectively in NLNRA and HLNRA individuals, which were not significantly different (t198 = 0.33; P = 0.739). The frequency of γH2AX foci was observed to be 0.090 ± 0.051, 0.096 ± 0.051, 0.076 ± 0.036, 0.087 ± 0.042, 0.108 ± 0.046 per cell, respectively in different dose groups of ≤ 1.50, 1.51-5.0, 5.01-10.0, 10.01-15.0, > 15.0mGy/year (ANOVA, F4,195 = 2.18, P = 0.072) and suggested non-linearity in dose response. The frequency of γH2AX foci was observed to be 0.098 ± 0.042, 0.078 ± 0.037, 0.084 ± 0.042, 0.099 ± 0.058, 0.097 ± 0.06 and 0.114 ± 0.033 per cell in the age groups of ≤ 29, 30-34, 35-39, 40-44, 45-49 and ≥ 50 years, respectively (ANOVA, F5,194 = 2.17, P = 0.059), which suggested marginal influence of age on the baseline of DSBs. Personal habits such as smoking (No v/s Yes: 0.092 ± 0.047 v/s 0.093 ± 0.048, t198 = 0.13; P = 0.895) and drinking alcohol (No v/s Yes: 0.096 ± 0.052 v/s 0.091 ± 0.045, t198 = 0.62; P = 0.538) did not show any influence on DSBs in the population. CONCLUSION: The present study did not show any increase in DSBs in different dose groups of HLNRA compared to NLNRA, however, it suggested a non-linear dose response between DNA DSBs and chronic low dose radiation.

Evaluation of the influence of chronic low-dose radiation on DNA repair gene polymorphisms [XRCC1, XRCC3, PRKDC (XRCC7), LIG1, NEIL1] in individuals from normal and high level natural radiation areas of Kerala Coast.

Background: Single Nucleotide Polymorphisms (SNPs) at DNA repair genes are considered as potential biomarkers of radio-sensitivity. The coastal belt of Kerala in south west India has a patchy distribution of monazite in its beach sand that contains Th-232 and its decay products. Thus, radiation levels in this area vary from <1.0mGy to 45.0mGy/year. The areas with external gamma radiation dose >1.5mGy/year are considered as High-Level Natural Radiation Areas (HLNRA) and ≤ 1.5mGy/year are Normal Level Natural Radiation Area (NLNRA).Objective: In the present study, an attempt was made to evaluate the influence of chronic low dose radiation exposure on DNA repair gene polymorphisms in NLNRA and HLNRA population of Kerala coast.Materials and methods: Genomic DNA was isolated from venous blood samples of 246 random, healthy individuals (NLNRA, N = 104; HLNRA, N = 142) and genotyping of five SNPs such as X-ray repair cross complementing 1(XRCC1 Arg399Gln), X-ray repair cross complementing 3 (XRCC3 Thr241Met], Protein kinase, DNA-activated, catalytic subunit (PRKDC) (X-ray repair cross-complementing group 7, XRCC7 G/T), nei like DNA glycosylase 1 (NEIL1 G/T) and DNA ligase 1 (LIG1 A/C) was carried out using PCR based restriction fragment length polymorphism (PCR-RFLP) followed by silver staining.Results: Our results showed no significant difference in genotype frequencies in HLNRA vs NLNRA at three of the five SNPs studied i.e. XRCC1 Arg399Gln (χ2(2) = 5.85, p = .054), XRCC3 Thr241Met (χ2(1) = 0.71, p = .339), PRKDC (XRCC7 G/T) (χ2(2) = 3.72, p = .156), whereas significant difference was observed at NEIL1 G/T (χ2(2) =8.71, p = .013) and LIG1 A/C (χ2(2) = 7.66, p = .022). The odds of heterozygote to homozygote genotypes in HLNRA relative to NLNRA at XRCC1 Arg399Gln (OR = 1.96, 95% CI: 1.13-3.40), XRCC3 Thr241Met (OR = 0.73, 95% CI: 0.41-1.31), PRKDC (XRCC7 G/T), (OR = 0.81; 95% CI: 0.48-1.38), NEIL1 G/T (OR = 0.54; 95% CI: 0.31-0.96) and LIG1 A/C (OR = 1.62; 95% CI: 0.97-2.69) was also not significantly different in HLNRA vs NLNRA, except at XRCC1 and NEIL1.Conclusion: The genotype frequencies at three of these SNPs i.e. XRCC1 Arg399Gln, XRCC3 Thr241Met and PRKDC (XRCC7 G/T) were similar, whereas NEIL1 G/T and LIG1 A/C showed significant difference between HLNRA and NLNRA population. However, further research using more number of SNPs in a larger cohort is required in this study area.

Lack of increased DNA double-strand breaks in peripheral blood mononuclear cells of individuals from high level natural radiation areas of Kerala coast in India.

The high level natural radiation area (HLNRA) of Kerala is a 55km long and 0.5km wide strip in south west coast of India. The level of background radiation in this area varies from <1.0mGy/year to 45.0mGy/year. It offers unique opportunity to study the effect of chronic low dose/low dose-rate radiation directly on human population. Spontaneous level of DNA double strand breaks (DSBs) was quantified in peripheral blood mononuclear cells of 91 random individuals from HLNRA (N=61, mean age: 36.1±7.43years) and normal level natural radiation area (NLNRA) (N=30, mean age: 35.5±6.35years) using gamma-H2AX as a marker. The mean annual dose received by NLNRA and HLNRA individuals was 1.28±0.086mGy/year and 8.28±4.96mGy/year, respectively. The spontaneous frequency of DSBs in terms of gamma-H2AX foci among NLNRA and HLNRA individuals were 0.095±0.009 and 0.084±0.004 per cell (P=0.22). The individuals from HLNRA were further classified as low dose group (LDG, 1.51-5.0mGy/year, mean dose: 2.63±0.76mGy/year) and high dose group (HDG, >5.0mGy/year, mean dose: 11.04±3.57mGy/year). The spontaneous frequency of gamma-H2AX foci per cell in NLNRA, LDG and HDG was observed to be 0.095±0.009, 0.096±0.008 and 0.078±0.004 respectively. Individuals belonging to HDG of HLNRA showed marginally lower frequency of DSBs as compared to NLNRA and LDG of HLNRA. This could be suggestive of either lower induction or better repair of DSBs in individuals from HDG of HLNRA. The present study indicated that 5.0mGy/year could be a possible threshold dose for DSB induction at chronic low-dose radiation exposure in vivo. However, further studies on DNA damage induction and repair kinetics are required to draw firm conclusions.

Peripheral blood lymphocyte micronucleus frequencies in men from areas of Kerala, India, with high vs normal levels of natural background ionizing radiation.

We have measured the frequencies of micronuclei (MN) in adult male individuals living in areas of the Kerala coast, southwest India, with either high (HLNRA, >1.5mGy/year) or normal levels of natural ionizing radiation (NLNRA, ≤1.5mGy/year). Blood samples were obtained from 141 individuals, 94 from HLNRA and 47 from NLNRA, aged 18-72, and were subjected to the cytokinesis-block micronucleus (CBMN) assay. An average of 1835 binucleated (BN) cells per individual were scored. The overall frequency of MN (mean±SD) was 11.7±6.7 per 1000 BN cells. The frequencies of MN in the HLNRA (11.7±6.6) and NLNRA (11.6±6.7) were not statistically significantly different (P=0.59). However, a statistically significant (P<0.001) age-dependent increase in MN frequency was observed among individuals from both HLNRA and NLNRA. No natural background radiation dose-dependent increase in MN frequency was seen. MN frequency was not influenced by tobacco smoking or chewing but it was increased among individuals consuming alcohol. Chronic low-dose radiation in the Kerala coast did not have a significant effect on MN frequency among adult men.

Study of stillbirth and major congenital anomaly among newborns in the high-level natural radiation areas of Kerala, India.

Monitoring newborns for adverse outcomes like stillbirth and major congenital anomalies (MCA) is being carried out in government hospitals since 1995 in and around high-level natural radiation areas, a narrow strip of land on the southwest coast of Kerala, India. Natural deposits of monazite sand containing thorium and its daughter products account for elevated levels of natural radiation. Among 141,540 newborns [140,558 deliveries: 139,589 singleton, 957 twins (6.81 ‰), 11 triplets (0.078 ‰), and one quadruplet] screened, 615 (4.35 ‰) were stillbirth and MCA were seen in 1,370 (9.68 ‰) newborns. Clubfoot (404, 2.85 ‰) was the most frequent MCA followed by hypospadias (152, 2.10 ‰ among male newborns), congenital heart disease (168, 1.19 ‰), cleft lip/palate (149, 1.05 ‰), Down syndrome (104, 0.73 ‰), and neural tube defects (72, 0.51 ‰). Newborns with MCA among stillbirths were about 20-fold higher at 190.24 ‰ (117/615) compared to 8.89 ‰ (1,253/140,925) among live births (P < .001). Logistic regression was carried out to compare stillbirth, overall, and specific MCA among newborns from areas with dose levels of ≤1.5, 1.51-3.0, 3.01-6.0 and >6 mGy/year after controlling for maternal age at birth, gravida, consanguinity, ethnicity, and gender of the baby. Clubfoot showed higher prevalence of 3.26 ‰ at dose level of 1.51-3.0 mGy/year compared to 2.33 ‰ at ≤1.5 mGy/year (OR = 1.39; 95 % CI, 1.12-1.72), without indication of any clear dose-response. Prevalences of stillbirth, overall MCA, and other specific MCA were similar across different dose levels and were relatively lower than that reported elsewhere in India, probably due to better literacy, health awareness, and practices in the study population.

Cytogenetic studies on newborns from high and normal level natural radiation areas of Kerala in southwest coast of India.

PURPOSE: To study, characterize and compare chromosome aberrations and karyotype anomalies among newborns from high (> 1.5 mGy/y) and normal (≤ 1.5 mGy/y) level natural radiation areas of monazite-sand bearing southwest coast of Kerala in India. MATERIALS AND METHODS: Cord blood samples from newborns were collected from selected Government hospitals in heparinized vials and cultures were set up employing standard microculture techniques, slides were prepared, coded and stained with giemsa. Well spread metaphases were analyzed for chromosome aberrations and karyotype anomalies. RESULTS: A total of 1,267,788 metaphases from 27,295 newborns of mothers aged 17-45 years (17,298 from high and 9,997 from normal level radiation areas) were analyzed during 1986-2007. Frequencies of dicentrics in high and normal level radiation areas were 1.90 ± 0.14 and 2.01 ± 0.26 per 10,000 cells, respectively (Relative frequency [RF] = 0.94; 95% CI: 0.71-1.26). Karyotype anomalies had a frequency of 5.49‰ and 6.7‰, respectively (RF = 0.82; 95% CI: 0.60-1.12). No dose-related trend was observed in chromosome aberrations or karyotype anomalies. CONCLUSION: Frequencies of chromosomal aberration and karyotype anomalies between the newborns from the high level natural radiation area (HLNRA) and normal level natural radiation areas (NLNRA) were very similar.

Effect of low and chronic radiation exposure: a case-control study of mental retardation and cleft lip/palate in the monazite-bearing coastal areas of southern Kerala.

A population-based 1:3 age-matched case-control study was conducted during 2006-2009 to assess the role of high-level natural radiation (>1 mSv/year) on congenital mental retardation and cleft lip/palate in the southwest coastal area of Kerala. Dosimetry was carried out in the house where parents resided during conception and the subsequent two trimesters of pregnancy of the study subject. Conditional logistic regression did not suggest any statistically significant association of either mental retardation (n = 445) or cleft lip/palate (n = 116) with high-level natural radiation. The odds of mental retardation and cleft lip/palate among those exposed to high-level natural radiation relative to normal levels of natural background radiation (≤1 mSv/year) were 1.26 (95% CI: 0.91-1.73) and 0.56 (95% CI: 0.31-1.02), respectively, after controlling for gender and maternal age at birth of the study subject. The data did not suggest any dose-related trend in the risk of either mental retardation (P = 0.113) or cleft lip/palate (P = 0.908). Notwithstanding the use of a single dose estimate to reconstruct past radiation exposure and the complex etiology of congenital malformations, it may reasonably be concluded that the prevailing high-level natural radiation in the study area does not appear to increase the risk of either mental retardation or cleft lip/palate among offspring of parents staying in the area.

Inhalation and external doses in coastal villages of high background radiation area in Kollam, India.

The observational evidence for radiation-induced health effects in humans comes largely from the exposures to high doses received over short periods of time. The rate of induction of any health risk at low doses and dose rates is estimated by extrapolation from observations at high doses. Effects of low dose/low dose rate could be done by the study of populations that have been exposed to slightly above-average natural radiation doses. Southwest coastal line of the Kerala state in India is one such region known to have elevated levels of background radioactivity mainly due to the mineral-rich sand available with high abundance of thorium. In the present work, a study was conducted to investigate the inhalation and external radiation doses to human beings in the high background radiation area along the southwest coast of Kerala. Five hundred dwellings were selected for the study. All the selected houses were at least 10 y old with similar construction. Long-term integrated indoor measurements of the external gamma dose using thermoluminescent dosemeters (TLDs) and the inhalation dose with the SSNTD-based twin-cup dosemeters were carried out in the dwellings simultaneously. Ambient gamma dose measurements were also made with a GM tube-based survey meter while deploying and retrieving the dosemeters. The data show a high degree of heterogeneity. The inhalation dose was found to vary from 0.1 to 3.53 mSv y(-1) and the external dose rates had a range of 383-11419 µGy y(-1). The external doses measured by the survey meter and TLDs showed an excellent correlation.

Deposition-based passive monitors for assigning radon, thoron inhalation doses for epidemiological studies.

The International Commission on Radiological Protection dose limits for radiation protection have been based on linearly extrapolating the high-dose risk coefficients obtained from the Japanese A bomb survivor data to low doses. The validity of these extrapolations has been questioned from time to time. To overcome this, epidemiological studies have been undertaken across the world on populations chronically exposed to low-radiation levels. In the past decade, the results of these studies have yielded widely differing, and sometimes, contradictory, conclusions. While recent residential radon studies have shown statistically significant radon risks at low doses, high-level natural radiation (HLNR) studies in China and India have not shown any low-dose risks. Similar is the case of a congenital malformation study conducted among the HLNR area populations in Kerala, India. It is thus necessary to make efforts at overcoming the uncertainties in epidemiological studies. In the context of HLNR studies, assigning radon and thoron doses has largely been an area of considerable uncertainty. Conventionally, dosimetry is carried out using radon concentration measurements, and doses have been assigned using assumed equilibrium factors for the progeny species. Gas-based dose assignment is somewhat inadequate due to variations in equilibrium factors and possibly due to significant thoron. In this context, passive, deposition-based progeny dosimetry appears to be a promising alternative method to assess inhalation doses directly. It has been deployed in various parts of India, including HBRAs and countries in Europe. This presentation discusses the method, the results obtained and their relevance to dose assignment in Indian epidemiological studies.