Gene hypermethylation in blood leukocytes in humans long term after radiation exposure - Validation set.

Hypermethylation of СpG islands in the promoter regions of several genes with basic protective function in blood leukocytes of individuals exposed to ionizing radiation long time ago (2-46 years), and differential effects of age and radiation exposure on hypermethylation was reported in our previous work. To validate these results, epigenetic modifications were assessed in an independent series of 49 nuclear industry workers from the "Mayak" facility (67-84 years old at sampling) with documented individual accumulated doses from the prolonged external γ-radiation exposure (95.9-409.5 cGy, end of work with radiation:0.3-39 years ago), and in 50 non-exposed persons matched by age. In addition to the genes analyzed before (RASSF1A, p16/INK4A, p14/ARF, GSTP1), four additional loci were analyzed: TP53, ATM, SOD3, ESR1. The frequency of individuals displaying promoter methylation of at least one of the 8 genes (71.4%) was significantly higher in exposed group as compared to the control group (40%), p = .002, OR = 3.75. A significantly elevated frequency of individuals with hypermethylated СpG islands in GSTP1, TP53, SOD3 promoters was revealed among exposed subjects as compared to the control group (p = .012, OR = 8.41; p = .041, OR = 4.02 and p = .009, OR = 3.42, respectively). A similar trend (p = .12, OR = 3.06) was observed for the p16/INK4A gene. As a whole, p16/INK4A and GSTP1 promoter hypermethylation in irradiated subjects from both previously and currently analyzed groups was pronounced. Thus, the direction of the effects was fully confirmed, suggesting the result reproducibility. No statistically significant correlation between promoter methylation and individual radiation dose was found. Further studies are required to create an array of blood epigenetic markers of radiation exposure associating with premature aging and age-related diseases and to accurately evaluate radiation-added effect across the range of doses. SYNTHESIS: The results of studies of epigenetic changes in two independent samples of irradiated subjects indicated the significance of radiation factor in the induction of hypermethylation of CpG islands in gene promoters that is revealed in blood cells years and decades after exposure.

Association of radiation-induced genes with noncancer chronic diseases in Mayak workers occupationally exposed to prolonged radiation.

We examined the association of gene expression with noncancer chronic disease outcomes in Mayak nuclear weapons plant workers who were exposed to radiation due to their occupation. We conducted a cross-sectional study with selection based on radiation exposure status of Mayak plant workers living in Ozyorsk who were alive in 2011 and either exposed to: combined incorporated Plutonium-239 ((239)Pu) and external gamma-ray exposure (n = 82); external gamma-ray exposure alone (n = 18); or were unexposed (n = 50) of Ozyorsk residents who provided community-based professional support for plant personnel and who were alive in 2011. Peripheral blood was taken and RNA was isolated and then converted into cDNA and stored at -20°C. In a previous analysis we screened the whole genome for radiation-associated candidate genes, and validated 15 mRNAs and 15 microRNAs using qRT-PCR. In the current analysis we examined the association of these genes with 15 different chronic diseases on 92 samples (47 males, 45 females). We examined the radiation-to-gene and gene-to-disease associations in statistical models stratified by gender and separately for each disease and exposure. We modeled radiation exposure as gamma or (239)Pu on both the continuous and categorical scales. Unconditional logistic regression was used to calculate odds ratios (OR), 95% confidence intervals (CI), and the concordance for genes that were significantly associated with radiation exposure and a specific disease outcome were identified. Altogether 12 mRNAs and 9 microRNAs appeared to be significantly associated with 6 diseases, including thyroid diseases (3 genes, OR: 1.2-5.1, concordance: 71-78%), atherosclerotic diseases (4 genes, OR: 2.5-10, concordance: 70-75%), kidney diseases (6 genes, OR: 1.3-8.6, concordance: 69-85%), cholelithiasis (3 genes, OR: 0.2-0.3, concordance: 74-75%), benign tumors [1 gene (AGAP4), OR: 3.7, concordance: 81%] and chronic radiation syndrome (4 genes, OR: 2.5-4.3, concordance: 70-99%). Further associations were found for systolic blood pressure (6 genes, OR: 3.7-10.6, concordance: 81-88%) and body mass index [1 gene (miR-484), OR: 3.7, concordance: 81%]. All associations were gender and exposure dependent. These findings suggest that gene expression changes observed after occupational prolonged radiation exposures may increase the risk for certain noncancer chronic diseases.

Independent validation of candidate genes identified after a whole genome screening on Mayak workers exposed to prolonged occupational radiation.

We evaluated gene expression in the peripheral blood of Mayak workers in relationship to occupational chronic exposure to identify permanent post-exposure signatures. The Mayak workers had experienced either a combined exposure to incorporated (239)Pu and external gamma rays (n = 82) or exposure to external gamma rays (n = 18). Fifty unexposed individuals served as controls. Peripheral blood was collected and then the RNA was isolated, converting it into cDNA and stored at -20°C. In a previous study at stage I, we screened the mRNA and microRNA transcriptome using 40 of the 150 samples and identified 95 mRNAs and 45 microRNAs. In stage II of this study, we now validated our 140 candidate genes using the qRT-PCR technique for the remaining 92 blood samples (18 samples were lost due to methodological reasons). We analyzed associations of normalized gene expression values in linear models separately for both exposure types (continuous and categorical scales) and adjusted for exposure age as well as stratified by gender. After further adjustment for confounders such as chronic non-cancer diseases or age at biosampling, mostly binary (on/off) dose-to-gene relationships were found for 15 mRNAs and 15 microRNAs, of which 8 mRNAs and 6 microRNAs remained significant after Bonferroni correction. Almost all of them were associated with plutonium incorporation and gender. Our study provides mRNA and microRNA gene expression changes dependent on the exposure type and gender, which occur and seem to persist after chronic radiation exposures supporting the concept of permanent post-exposure signatures.

Gene expression analysis in Mayak workers with prolonged occupational radiation exposure.

The authors evaluated gene expression in the peripheral blood in relation to occupational exposure in Mayak workers to find out about the existence of a permanent post exposure signature. Workers were exposed to combined incorporated ²³9Pu and external gamma rays (n = 82) or to external gamma rays only (n = 18), and 50 unexposed individuals served as controls. Peripheral blood was taken from workers older than 70 y. RNA was isolated, converted into cDNA, and stored at -20°C. A two-stage study design was performed focusing on examinations on the transcriptional (mRNA) and post-transcriptional level (microRNA). In the first stage, 40 samples were identified for screening purposes and selection of candidate genes. For examinations on the transcriptional level, whole genome microarrays and qRT-PCR were employed on the post-transcriptional level (667 microRNAs). Candidate genes were assessed by (1) introducing a twofold difference in gene expression over the reference group and (2) showing a significant p-value using the Kruskal-Wallis test. From 42,545 transcripts of the whole genome microarray, 376 candidate genes (80 up-regulated and 296 down-regulated relative to the reference group) were selected. Expression of almost all of these genes (70-98%) appeared significantly associated with internal ²³9Pu and to a lesser extent were associated with external gamma-ray exposure (2-30%). Associations in the same direction were found for 45 microRNAs. Although both exposures led to modulations of different gene sets in different directions, the authors could detect no differences in gene set enrichment analysis.

Radiation-induced lung adenocarcinoma is associated with increased frequency of genes inactivated by promoter hypermethylation.

Epigenetic inactivation of genes by promoter hypermethylation, a major mechanism in the initiation and progression of tobacco-induced cancer, has also been associated with lung cancer induced through environmental and occupational exposures. Our previous study of gene methylation in workers from the MAYAK nuclear enterprise identified a significantly higher prevalence for methylation of the p16 gene (CDKN2A) in adenocarcinomas from workers compared to tumors from non-worker controls. The purpose of this investigation was to determine whether genes in addition to p16 are "targeted" for silencing and whether overall gene methylation was more common in radiation-induced adenocarcinoma. A significant increase in the prevalence of methylation of GATA5 was seen in tumors from workers compared to tumors from controls. The prevalence for methylation of PAX5 beta and H-cadherin did not differ in tumors from workers and controls. Evaluating the frequency for methylation of a five-gene panel revealed that 93% of adenocarcinomas from workers compared to 66% of tumors from controls were methylated for at least one gene. Moreover, a twofold increase was seen in the number of tumors methylated for three or more genes for tumors from workers compared to controls. Increased frequency for inactivation of genes by promoter hypermethylation and targeting of tumor suppressor genes such as GATA5 may be factors that contribute to the increased risk for lung cancer associated with radiation exposure.

Plutonium targets the p16 gene for inactivation by promoter hypermethylation in human lung adenocarcinoma.

Lung cancer from radon or (239)plutonium exposure has been linked to alpha-particles that damage DNA through large deletions and point mutations. We investigated the involvement of an epigenetic mechanism, gene inactivation by promoter hypermethylation in adenocarcinomas from plutonium-exposed workers at MAYAK, the first Russian nuclear enterprise established to manufacture weapons plutonium. Adenocarcinomas were collected retrospectively from 71 workers and 69 non-worker controls. Lung adenocarcinomas were examined from workers and non-worker controls for methylation of the CDKN2A (p16), O(6)-methylguanine-DNA methyltransferase (MGMT), death associated protein kinase (DAP-K), and Ras effector homolog 1 genes (RASSF1A). The prevalence for methylation of the MGMT or DAP-K genes did not differ between workers and controls, while a higher prevalence for methylation of the RASSF1A gene was seen in tumors from controls. In marked contrast, the prevalence for methylation of p16, a key regulator of the cell cycle, was increased significantly (P = 0.03) in tumors from workers compared with non-worker controls. Stratification of plutonium exposure into tertiles also revealed a striking dose response for methylation of the p16 gene (P = 0.008). Workers in the plutonium plant where exposure to internal radiation was highest had a 3.5 times (C.I. 1.5, 8.5; P = 0.001) greater risk for p16 methylation in their tumors than controls. This increased probability for methylation approximated the 4-fold increase in relative risk for adenocarcinoma in this group of workers exposed to plutonium. In addition, a trend (P = 0.08) was seen for an increase in the number of genes methylated (> or =2 genes) with plutonium dose. Here we demonstrate that exposure to plutonium may elevate the risk for adenocarcinoma through specifically targeting the p16 gene for inactivation by promoter methylation.