Cytogenetic and Gene Expression Study in Radiation Workers Occupationally Exposed to Low Levels of Ionizing Radiation

Background: Exposure to IR (ionizing radiation) causes damage to living cells, especially to DNA, the degree of cellular damage depends on the radiation amount. Humans are naturally exposed to IR from cosmic rays, and artificially through diagnostic procedures, medical treatments or occupationally during work shifts. Cytogenetic assay of peripheral blood lymphocytes can provide a biological estimation of the dose received in IR exposures. The CBMN (cytokinesis-blocked micronucleus) assay is a widely used, since it represents a reliable test to detect radiation-induced chromosome aberration and it is a valuable biomarker in many biomonitoring studies on human populations occupationally exposed to IR. The NDI (nuclear division index) is a cell proliferation marker in cultures which is considered a measure of general cytotoxicity, the relative frequencies of the cells may be used to define cell cycles progression of the lymphocyte after mitogenic stimulation. Nevertheless the assay is frequently employed as a useful research tool for understanding the cell cycling kinetics of the cultures. Many gene expression studies demonstrated an up-regulation of genes involved in the processes of signal transduction, control of cell cycle, DNA repair and apoptosis after exposure of IR in different mammalian cell types. Altered expression of a few genes plays specific roles in DNA repair/cell cycle control such as CDKN1A, XPC, GADD45A, DDB2 and PCNA and cell cycle regulation/proliferation such as IL16, CABLES2, TGFB2 and RHOA. Objective: The present study aims to use the MN, NDI and gene expression analysis as biomarkers for investigation of the effects of IR exposure in some radiation workers occupationally exposed to low IR in Al-Tuwaitha site, Baghdad, Iraq. Also, authors assessed the effect of IR on the expression of some gene marker such as: RHOA, CDKN1A, GADD45A and RAD52. Methods: This study was carried out on thirty Iraqi male radiation workers occupationally exposed to low levels of IR at Al-Tuwaitha site, Baghdad, non-smokers and non-alcoholic, aged 30-59 years, as well as thirty apparently healthy individuals males collected randomly from population living in Baghdad, aged 30-59 years who are non-smokers, non-alcoholic as control group. Three molecular genetic parameters were employed such as CBMN, NDI and gene expression assay. The MN and NDI assay were performed according to the description by IAEA, 2001. Total RNA was isolated from blood for radiation worker and control group. The RNA concentration was determined by measuring their absorbance that depends on the ratio A260/A280 of the wavelength, which leads to the determination of RNA purity, which ranged from 1.79-2.1 in two groups. Using RT-PCR for study gene expression, four types of specialized primer genes were selected for the mRNA genes: RHOA, CDKN1A, GADD45A and RAD52 which have a relation with IR in addition to the primers for internal control. Housekeeping gene ((β-actin) was used as a reference gene to normalize the quantity of the target genes. Results: The results of cytogenetic analysis showed that micronuclei frequencies were significantly higher (p < 0.01) in the radiation workers group in Al-Tuwaithasite, Baghdadas compared with the control group, and showed significant decrease (p > 0.01) in the NDI in radiation workers as compared with the control group. Total RNA was isolated from blood for the radiation workers and control groups mentioned. Using RT-PCR for study gene expression, four types of specialized primer genes were selected for the mRNA: genes RHOA, CDKN1A, GADD45A and RAD52 which have a relation with IR in addition to the primers for internal control (β-actin) gene. The products of replicated specialized primers for the genes concerned and the cDNA for the studied samples were electrophoretically separated in agarose gels. The banding profiles were visualized by ethidium bromide staining, as the molecular weight were 135 bp, 165 bp, 185 bp and 470 bp (nitrogen-base pair) for RHOA, CDKN1A, GADD45A and RAD52 genes, respectively. Gene expression analysis revealed statistically significant (∆∆Ct comparative Ct method) transcriptional changes in three genes RHOA, GADD45A, CDKN1A up-regulated while the RAD52 gene down-regulated. Conclusions: The results indicated that there is a possibility of using the changes in the MN, NDI and genes expression such as RHOA, CDKN1A, GADD45A and RAD52 as useful biomarkers for detection of IR in radiation workers occupationally exposed to low levels of IR.