Alpha-particle-infuced DNA Double Strand Breaks And Their Repair Protein Expression Changes In Human Blood Lymphocyte

Radioactive nuclide especially high LET alpha radiator internal irradiation in the radiation hazards to human health occupies an extreme important position. So far to use biological indicator to judge whether the body were irradiated by alpha nuclide and its internal radiation of biological dose estimation problem, have yet to find the effective solutions at home and abroad. Currently phosphorylation of histone family 2A variant(γH2AX), which has become a recognized molecular markers of DNA double-strand break(DSBs), can recruit a variety of repair related proteins (such as participation in NHEJ of pDNA-PKcs(Thr2609) and HR of Rad51) to quickly gathered in injured part, forming Ionizing radiation-induced foci(IRIF), which analysis technics has become a sensitive method for monitoring DSBs and its repair; The key choice factor of DSBs repair pathways of 53BP1 also is a major concern, whether γH2AX and 53BP1 IRIF can be the biological dose estimation index has become a research hotspot in recent years, but there are problems such as foci yield falling fast following the time prolonging after exposed to irradiation. Research has confirmed that the complexity of the damage and the distribution in the heterochromatin(HC) of DSB-cluster induced by high LET radiation are the important factors influencing the DSBs repair rate. This study observed the formation and loss of γH2AX,53BP1, pDNA-PKcs(Thr2609) and Rad51 IRIF induced by high LET α-particle irradiation and their spatial localization related with chromatin structure in the G0 lymphocytes of human peripheral blood measured by immunofluorescence staining technique, meanwhile detect the apoptosis rate measured by TUNEL-FITC method, compared with low LET γ-rays, for deep understanding repair characteristics of DSB-cluster damage induced by high LET radiation in the G0 lymphocytes of human peripheral blood has important significance and provide the experimental basis for the judgement and biological dose estimation of α-particle internal irradiation. This experiment also measured by immunofluorescence technique detect the expression of γH2AX,53BP1, pDNA-PKcs(T2609) and Rad51 IRIF in peripheral blood lymphocytes of radioactive workers and their relationship with chromosome aberration, in order to preliminary realize the expression level of four types of IRIF in the human body and estimate whether the radioactive workers have the recent internal exposurey, for searching the new indicators of assessment of ionization radiation hazards to he.alth and offer guidance for radiation protection and safety.Objective To investigate the characteristics of repair of DNA DSBs induced by high-LET a-particles irradiation and their relationship with chromatin structure in the Go lymphocytes of human peripheral blood, in order to provide the experimental basis for the judgement and dose estimation of a-particles internal irradiation.Methods Samples of peripheral whole blood were collected from four healthy volunteers and lymphocytes were separated, then lymphocytes were divided into the normal group, the 0.5 Gy a-particle irradiation group and the 0.5 Gy y-ray irradiation group. The formation and loss of yH2AX IRIF as a surrogate marker of DSB,53BP1 IRIF as DSB reaction protein, pDNA-PKcs(Thr2609) IRIF as a marker of NHEJ repair and Rad51 IRIF as a marker of HR repair and their spatial localization related with chromatin structure were measured by immunofluorescence staining technique at 10 min-48 h post-irradiation; The apoptosis rate of lymphocytes were measured by TUNEL-FITC method.Results The formation of linear yH2AX,53BPI and pDNA-PKcs(Thr2609) IRIF tracks were observe at 10 min-2 h post-irradiation in lymphocytes exposed to a-particle radiation, especially the yH2AX linear tracks show continuous and discontinuous linear morphological characteristics; yH2AX and 53BP1 linear tracks quickly reach peak at 10 min post-irradiation (P<0.001, P<0.001), pDNA-PKcs(Thr2609) linear tracks reach peak at 30 min post-irradiation (P<0.001), and the IRIF tracks almost completely disappeared at 6 h post-irradiation, there is not linear Rad51 IRIF tracks observed. But no linear yH2AX,53BP1, pDNA-PKcs(Thr2609) and Rad51 IRIF tracks were observed at 10 min-48 h after y-ray irradiation. The result of dynamics of formation and loss and their localization in chromatin display, the frequencies of yH2AX、53BP1 and pDNA-PKcs(Thr2609) IRIF peaked at 30 min after a-particle radiation (P<0.001, P<0.01 and P<0.001) and then decreased obviously during 6 h post-irradiation (P<0.001, P<0.05 and P<0.001), but the average number of residual yH2AX IRIF remained at approximately 16% at 24-48 h post-irradiation, but 53BP1 and pDNA-PKcs(Thr2609) IRIF fell to the bottom level. Moreover,27% of yH2AX IRIF located at DAPI-bright heterochromatin region at 10 min after a-particle radiation, suggesting that efficacy of DSBs repair may be decreased, but 53BP1 and pDNA-PKcs(Thr2609) IRIF located at DAPI-weak euchromatin region. In comparison, yH2AX、53BP1 and pDNA-PKcs(Thr2609) IRIF diffused randomly in nucleus predominantly located in DAPI-weak euchromatin region at 10 min-48 h after y-ray irradiation. The formation of yH2AX IRIF is significantly greater than the 53BP1 and pDNA-PKcs(Thr2609) IRIF after a-particle radiation at 30 min-6 h; the formation of 53BP1 IRIF is significantly greater than the pDNA-PKcs(Thr2609) IRIF, alpha particle radiation induced such effect was significantly higher than the effect of y-rays, which reminder that high LET radiation induced more DSBs small fragments than the low LET radiation to interference DSBs repair, so molecular markers of DSBs of yH2AX IRIF is a significant increase, recruitment of 53BP1 IRIF significantly reduced, pDNA-PKcs(Thr2609) IRIF representing only NHEJ repair pathways are activated, which significantly lower than 53BP1 IRIF. During 30 min-2 h after a-particle and y-ray irradiation, the frequencies of Rad51 IRIF slightly but not significantly increased as compared to background level, and the frequencies of co-localization of Rad51 foci and yH2AX foci were only 3%-8%.0.5 Gy a-particle irradiation induced higher apoptosis rate compared with y-ray irradiation at 24-48 h post-irradiation, Showing that high LET a-particles damage is more serious and are more likely to lead to cell death compared with low LET y-rays damage.Conclusions The formation of linear yH2AX,53BP1 and pDNA-PKcs(Thr2609) IRIF tracks, especially the yH2AX linear tracks show continuous and discontinuous linear morphological characteristics, induced by high-LET a-particle irradiation in Go human lymphocytes could be used as biological indicator to estimate whether a person has been exposed to α-particle internal irradiation. Prolonged Stable persistence of residual γH2AX IRIF has the feasibility for biological dose estimation index; NHEJ repair pathways play an important role in repairing of radiation induced DSBs in human lymphocytes.Part II Detection of DNA double strand breaks and their repair protein expression in human lymphocytes of radioactive workersObjective To investigate the expression of DSBs and their repair proteins IRIF in peripheral blood lymphocytes of radioactive workers and their relationship with chromosome aberration, in order to provide important basis for evaluation of the radiation damage of radioactive workers and offer guidance for radiation protection and safety.Methods Samples of peripheral whole blood were collected from seven non-radioactive workers and thirteen radioactive workers, and then lymphocytes were separated. The expression of yH2AX、53BP、pDNA-PKcs(Thr2609) and Rad51 IRIF were measured by immunofluorescence staining technique. Meanwhile detect chromosome aberration rate of human peripheral blood lymphocytes.Results The formation of of yH2AX,53BP1 and pDNA-PKcs (Thr2609) IRIF of in human lymphocytes of radioactive workers were no significant difference with non-radioactive workers. The frequencies of yH2AX IRIF within the scope of 0-0.03/cell are very low in two groups of people, the level of 53BP1 IRIF within the scope of 0.04-0.31/cell is relatively high, and can also detect the formation of pDNA-PKcs(Thr2609) IRIF, but the Rad51 IRIF is 0, the results suggest that the antibodies of 53BP1 and pDNA-PKcs(Thr2609) may be a nonspecific combination. The frequencies of of yH2AX,53BP1 and pDNA-PKcs(Thr2609) IRIF were no s ignificant difference related with gender in the two groups. The formation of yH2AX, 53BP1 and pDNA-PKcs(Thr2609) IRIF were no significant difference between radioactive workers more than 30 years old and non-radioactive workers less than 30 years old. One radioactive worker was the 0.5% of chromosome aberration rate, but there were no change of yH2AX,53BP1, pDNA-PKcs(Thr2609) and Rad51 IRIF.Conclusions The formation of yH2AX,53BP1 and pDNA-PKcs(Thr2609) and Rad51 IRIF were no significant difference between radioactive workers and non-radioactive workers, moreover, there were no certain correlation with chromosome aberration, age and sex, especially the frequencies of yH2AX IRIF are very low, that has the feasibility for the evaluation index of radiation injury, but it remains to be further research by increasing the sample size.