Investigation Of The Relationship Between DNA Repair-related Protein Gene Expression And Radiation Exposure Doses

With the application of nuclear energy and radiation energy more widely, people pay more attention to the radiation effects in environment and human health. So it is necessary to study bioeffects and develop process of the effects induced by radiation in further On the other hand, the establishment of new bio-method which can be used to assess radiation effects, diagnose and cure kinds of radiation induced injuries is very necessary now. As physical methods have many shortcomings and limits when used to evaluate exposure dose, how to use bioanalysis which may screen out kinds of biomarkers to assess exposure dose has been one of the most hot in the filds of radiobiology, radiation dosimetry and radiation protection. Lymphocyte dynamics and the dicentric assay are considered as the gold standard for radiation biodosimetry, however, practice shows that when exposed to certain dose peripheral lymphocyte count decreased rapidly to zero, so this indication method has dose range limit. When indicating exposure dose using chromosoral aberrations, the division ability and cell number in division phase of lymphocyte declining as increasing radiation dose is hard to ensure the accuracy and reliability of results beyond a certain dose ranges. In addition, chromosome analysis requires specialized laboratories, technicians and at least 48-72 hours, not suitable fot on-site rapid analysis.Biological methods can be used to assess the personnel exposure dose by analyzing the change of indicators related to radiation effects. As the change of indicators is the direct or indirect reflection of the radiation energy depositing in the body, making the analysis less dependent on the restrictions from radiation conditions and environment like physic method did, so the result can be more faithful and representative. And also it is easy to collect biological samples, so it can be used to facilitate rapid analysis of high throughput. For these reasons, biological method has been recognized as the preferred method internationally in the sudden nuclear radiological accidents when a large number of people needed for rapid screening of radiation doses. Screening and rational using biological indicators, the establishment of specific, sensitive and reliable methods of analysis are two areas of crucial importance when using Bioanalytical methods to assess exposure dose. With the development of new theories, new methods and the use of new technologies, a number of biomarkers which have the indicating exposure dose potential in levels of gene and protein are discovered and reported[1-2]. ATM (Ataxia Telangiectasia Mutated) is an important protein which can respond and pass DNA DSB damage signal and also induce a series functional proteins,such as H2AX, RAD50, NBS1, Mre11, BRCA1 and so on to repair DNA damage[3]. It is reported in 2000 in normal human skin fibroblasts the expression of BRCA1 had good relations with the radiation dose, when the radiation doses were 5, 10, 20 Gy, BRCA1 expression were 0.33, 0.38 and 0.44 respectively[4]. Recently reported, a potential link exist between the activation of H2AX and ATM protein and radiation dose, the study found the signal of ATM activation could be detected at 30 minutes post radiation with 0.25 Gy, and high fluorescent focus (foci) of the protein Activation could be detected at 3 hours after exposure with 4Gy[5-6]. As the fact that exposure dose has a logical connection with the degree of DNA damage and the expression of DNA repair related proteins, so it is very likely to obtain a set of indicators to characterize radiation dose information when analyzing multiple biomarkers in levels of gene, protein expression and activation. There is rich information related to radiation dose in DNA damage repair-related protein expression and activation, using specific and sensitive methods to detect and analyze such information, consolidation or combination the relationship between the information and dose is expected to filter out new suitable biological indicators to indicate exposure doses. The present study was to study the transcription expression change of a total of 11 DNA repair related genes which are ATM, BBC3, XPC, PLK3, RAD50, DDB2, FDXR, GADD45A, BRCA1, IER5 and CDKN1A and the translation expression change of P21, P53 and HSP70 in different post time points with different radiation dose in blood cells of TBI-treated BALB/c mice. By this study It will be helpful for constructing appropriate gene expression biomarker systems to evaluate radiation exposure doses.Methods1. Animal model and sample preparation:Male BALB/c mice, 6–8 weeks old were divided into control (non-irradiated) and four irradiated groups (six mice for each group). The irradiation groups received 2, 4, 6 or 8 Gy TBI at 0.80 Gy/min and 1.60 Gy/min with a ⁶⁰Coγ-ray source (Shanghai Institute of Measurement and Testing Technology, SIMT China) respectively. The source provided a 98% uniform exposure in 15 cm×13 cm.exposure field. The field uniformity was measured with a graphite cavity ionization chamber.2. Gene expression analysis:(1) Heparin-anticoagulated blood was collected from the orbital venous vein which were used to extract RNA used Axygen Blood Total RNA Miniprep kit (Axygen Scientific, America)(2) Real-time PCR: Using the SYBR Prime Script RT Reagent Kit, No. DRR037A (Takara, Dalian, China), 1 ?g of total RNA was reverse transcribed to cDNA according to the manufacturer's instructions. The RT-PCR reactions were performed with the Rotor-Gene 6000 RT-PCR System using the SYBR Premix Ex Taq, No. DRR041A (Takara, Dalian, China) following the manufacturer's recommendations.3. Protein expression analysis:(1) Heparin-anticoagulated blood was collected from the orbital venous vein which was used to extract protein.(2) Western blot method was used for detecting the changes of P21,P53 and HSP70 expression change.ResultsWithin 48 hours, the expression change of 11 DNA repair-related genes was examined after radiation, CDKN1A (cyclin-dependent kinase inhibitor 1A or p21, Cip1) and ATM (ataxia telangiectasia mutated) expression levels were heavily up- and downregulated, respectively, with increasing exposure doses at different post-irradiation time points. RAD50 (RAD50 homolog), PLK3 (polo-like kinase 3), GADD45A (growth arrest and DNA damage-inducible, alpha), DDB2 (damage-specific DNA-binding protein 2), BBC3 (BCL2 binding component 3) and IER5 (immediate early response 5) gene expression levels were found to undergo significant oscillating changes over a broad dose range of 2-8 Gy and at different post-exposure time points. These expression signatures can be combined as several groups to indicate exposure doses together. However accuracy and reliability in exposure dose estimation by these information still needs further experimental verification. The expression of proteins p53 and HSP70 in irradiated groups increased significantly compared to the control, but no significant change between irradiated groups. was found. Unfortunately the bands of P21 weren't gotten in this experiment.ConclusionsGene expression signatures associated with DNA damage and repair, especially those of the CDKN1A and ATM genes, in the blood cells of total body irradiated mice enrich the biomarker data pool for biodosimetry and will be helpful for constructing appropriate gene expression biomarker systems to evaluate radiation exposure doses. As a semi-quantitative protein analysis method Western Blot may act as a primary tool in protein biomarkers screening. More studies should be done to investigate expression of DNA repair-related proteins in order to find suitable protein biomarkers with new fast and accurate methods.