Telomere length in Chernobyl accident recovery workers in the late period after the disaster

摘要

Study participants were randomly selected from the CNPP clean-up workers examined in the Centre of Occupational and Radiological Medicine Inpatient and Outpatient Departments of the Pauls Stradins Clinical University Hospital (‘the Centre') in Riga. The selection criteria for exposed participants were people: (i) who have survived at least 20 years after participation in clean-up works, (ii) for whom there is documented and proven participation in CNPP clean-up works at any time during the period from 26 April 1986 till the end of 1991, (iii) with known data about current health condition, and (iv) who voluntarily participated and provided signed, informed written consent. Exclusion criteria were: (i) refusal of voluntary participation in the study, and (ii) people with extremely severe somatic pathology preventing them from coming to the Centre. All study participants were males. Most of these men participated in CNPP clean-up works for a period of 1–3 months. The group of CNPP clean-up workers studied was a subset of the 6000 Latvian inhabitants sent by the Soviet Union to Chernobyl to combat the consequences of the disaster. Blood samples were collected from CNPP clean-up workers between 2010 and 2012, i.e. ～23 years (range, 19–26 years) after their work in Chernobyl during the period 1986–1991. Blood samples were obtained from the cubital vein in 3-ml sterile vacutainers coated with EDTA (ethylenediaminetetraacetic acid). Vacutainers were labelled and immediately transported to the laboratory within 24 h. Genomic DNA was isolated from whole blood using a FlexiGene Kit (205) (Qiagen, Dusseldorf, Germany) according to the manufacturer's instructions. Acquired DNA quality was tested by 1% agarose gel electrophoresis. DNA concentration was measured by NanoDrop Spectrophotometer ND-1000. DNA samples were stored in 10 mM Tris-Cl (tris(hydroxymethyl)amino methane) buffer solution at pH = 8.5 and +4°C temperature until the time of assay. RTL was measured by real-time quantitative polymerase chain reaction (q-PCR) using the approach described by Cawthon [36]. DNA samples were diluted to a concentration of 10.0 ng/μl prior to performing real-time q-PCR. For reference, a DNA sample from a healthy age-matched male without malignancies in anamnesis and not previously exposed to excessive ionizing radiation (other than natural background radiation and rare small medical X-ray examinations) was selected. A dilution line of concentrations 50.0 ng/μl, 16.8 ng/μl, 5.25 ng/μl and 1.82 ng/μl was made from the reference DNA sample. Reference samples were amplified in each run. The PCR mixture was prepared as follows: 2.5 μl of × 10 Taq polymerase buffer (Takara, Japan), 0.2 μl of 10 mM dNTP mixture (Takara, Japan), 2.25 μl of em1109 primer (10 pM), 2.25 μl of em1110 primer (10 pM), 2.25 μl of em1111 primer (10 pM), 2.25 μl of em1112 (10 pM) primer, 2 μl of 5 nM SYBR Green (Invitrogen, USA), 0.5 U of Taq polymerase (SPEED STAR, Takara, Japan) and 9.3 μl of distilled water. To each well, 2 μl of diluted DNA sample and 23 μl of PCR mixture were added. The specific telomere and single-copy gene primers were supplied by Integrated DNA Technologies (IDT), Inc., USA. The primer sequences were as follows: Appropriate statistical methods were employed according to the shape of data distribution. The models were adjusted for age. For achieving precise age-effect evaluation, data were weighted by age before analysis using an integrated weighting approach. For performing detailed analysis, outliers were excluded from calculations. The association of RTL with age was evaluated using Spearman's correlation analysis. For comparing subgroups with each other, independent samples t-tests and Mann–Whitney tests were used, and odds ratios (ORs) and the 95% confidence intervals (CIs) were calculated. The significance level was set at 0.05. All calculations were completed using Microsoft Excel and IBM SPSS Statistics Version 20.