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An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...

University of the Western Cape

The lack of information on how biological systems respond to low-dose and low dose-rate exposures makes it difficult to accurately assess the carcinogenic risks. This is of critical importance to space radiation, which remains a serious concern for long-term manned space exploration. In this study, the γ-H2AX foci assay was used to follow DNA double-strand break (DSB) induction and repair following exposure to neutron irradiation, which is produced as secondary radiation in the space environment. Human lymphocytes were exposed to high dose-rate (HDR: 0.400 Gy/min) and low dose-rate (LDR: 0.015 Gy/min) (66)/Be(40) neutrons. DNA DSB induction was investigated 30 min post exposure to neutron doses ranging from 0.125 to 2 Gy. Repair kinetics was studied at different time points after a 1 Gy neutron dose. Our results indicated that γ-H2AX foci formation was 40% higher at HDR exposure compared to LDR exposure. The maximum γ-H2AX foci levels decreased gradually to 1.65 ± 0.64 foci/cell (LDR) and 1.29 ± 0.45 (HDR) at 24 h postirradiation, remaining significantly higher than background levels. This illustrates a significant effect of dose rate on neutron-induced DNA damage. While no significant difference was observed in residual DNA damage after 24 h, the DSB repair half-life of LDR exposure was slower than that of HDR exposure. The results give a first indication that the dose rate should be taken into account for cancer risk estimations related to neutrons.

The Impact of Dose Rate on DNA Double-Strand Break Formation and Repair in Human Lymphocytes Exposed to Fast Neutron Irradiation.

The number of proton therapy facilities and the clinical usage of high energy proton beams for cancer treatment has substantially increased over the last decade. This is mainly due to the superior dose distribution of proton beams resulting in a reduction of side effects and a lower integral dose compared to conventional X-ray radiotherapy. More recently, the usage of metallic nanoparticles as radiosensitizers to enhance radiotherapy is receiving growing attention. While this strategy was originally intended for X-ray radiotherapy, there is currently a small number of experimental studies indicating promising results for proton therapy. However, most of these studies used low proton energies, which are less applicable to clinical practice; and very small gold nanoparticles (AuNPs). Therefore, this proof of principle study evaluates the radiosensitization effect of larger AuNPs in combination with a 200 MeV proton beam. CHO-K1 cells were exposed to a concentration of 10 μg/ml of 50 nm AuNPs for 4 hours before irradiation with a clinical proton beam at NRF iThemba LABS. AuNP internalization was confirmed by inductively coupled mass spectrometry and transmission electron microscopy, showing a random distribution of AuNPs throughout the cytoplasm of the cells and even some close localization to the nuclear membrane. The combined exposure to AuNPs and protons resulted in an increase in cell killing, which was 27.1% at 2 Gy and 43.8% at 6 Gy, compared to proton irradiation alone, illustrating the radiosensitizing potential of AuNPs. Additionally, cells were irradiated at different positions along the proton depth-dose curve to investigate the LET-dependence of AuNP radiosensitization. An increase in cytogenetic damage was observed at all depths for the combined treatment compared to protons alone, but no incremental increase with LET could be determined. In conclusion, this study confirms the potential of 50 nm AuNPs to increase the therapeutic efficacy of proton therapy.

Radiosensitization Effect of Gold Nanoparticles in Proton Therapy.

The radiosensitivity of haematopoietic stem and progenitor cells (HSPCs) to neutron radiation remains largely underexplored, notwithstanding their potential role as target cells for radiation-induced leukemogenesis. New insights are required for radiation protection purposes, particularly for aviation, space missions, nuclear accidents and even particle therapy. In this study, HSPCs (CD34CD38 cells) were isolated from umbilical cord blood and irradiated with Co γ-rays (photons) and high energy p(66)/Be(40) neutrons. At 2 h post-irradiation, a significantly higher number of 1.28 ± 0.12 γ-H2AX foci/cell was observed after 0.5 Gy neutrons compared to 0.84 ± 0.14 foci/cell for photons, but this decreased to similar levels for both radiation qualities after 18 h. However, a significant difference in late apoptosis was observed with Annexin-V/PI assay between photon and neutron irradiation at 18 h, 43.17 ± 6.10% versus 55.55 ± 4.87%, respectively. A significant increase in MN frequency was observed after both 0.5 and 1 Gy neutron irradiation compared to photons illustrating higher levels of neutron-induced cytogenetic damage, while there was no difference in the nuclear division index between both radiation qualities. The results point towards a higher induction of DNA damage after neutron irradiation in HSPCs followed by error-prone DNA repair, which contributes to genomic instability and a higher risk of leukemogenesis.

Monique Engelbrecht

Department of Medical Biosciences

University of the Western Cape

An Automated Microscopic Scoring Method for the γ-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Monique Engelbrecht

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Medical Biosciences

University of the Western Cape

An Automated Microscopic Scoring Method for the γ-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Department of Medical Biosciences