BackgroundThere is a crucial shortage of methods capable of determining the extent of accidental exposures of human beings to ionizing radiation. However, knowledge of individual exposures is essential for early triage during radiological incidents to provide optimum possible life-sparing medical procedures to each person.Methods and FindingsWe evaluated immunocytofluorescence-based quantitation of γ-H2AX foci as a biodosimeter of total-body radiation exposure (60Co γ-rays) in a rhesus macaque (Macaca mulatta) model. Peripheral blood lymphocytes and plucked hairs were collected from 4 cohorts of macaques receiving total body irradiation doses ranging from 1 Gy to 8.5 Gy. Each cohort consisted of 6 experimental and 2 control animals. Numbers of residual γ-H2AX foci were proportional to initial irradiation doses and statistically significant responses were obtained until 1 day after 1 Gy, 4 days after 3.5 and 6.5 Gy, and 14 days after 8.5 Gy in lymphocytes and until 1 day after 1 Gy, at least 2 days after 3.5 and 6.5 Gy, and 9 days after 8.5 Gy in plucked hairs.ConclusionThese findings indicate that quantitation of γ-H2AX foci may make a robust biodosimeter for analyzing total-body exposure to ionizing radiation in humans. This tool would help clinicians prescribe appropriate types of medical intervention for optimal individual outcome. These results also demonstrate that the use of a high throughput γ-H2AX biodosimeter would be useful for days post-exposure in applications like large-scale radiological events or radiation therapy. In addition, this study validates a possibility to use plucked hair in future clinical trials investigating genotoxic effects of drugs and radiation treatments.

Measurement of DNA double-strand break (DSB) levels in cells is useful in many research areas, including those related to DNA damage and repair, tumorigenesis, anti-cancer drug development, apoptosis, radiobiology, environmental effects, and aging, as well as in the clinic. DSBs can be detected in the nuclei of cultured cells and tissues with an antibody to H2AX phosphorylated on serine residue 139 (γ-H2AX). DSB levels can be obtained either by measuring overall γ-H2AX protein levels in a cell population or by counting γ-H2AX foci in individual nuclei. Total levels can be obtained in extracts of cell populations by immunoblot analysis, and in cell populations by flow cytometry. Furthermore, with flow cytometry, the cell cycle distribution of a population can be obtained in addition to DSB levels, which is an advantage when studying anti-cancer drugs targeting replicating tumor cells. These described methods are used in genotoxicity assays of compounds of interest or in analyzing DSB repair after exposure to drugs or radiation. Immunocyto/immunohistochemical analysis can detect γ-H2AX foci in individual cells and is very sensitive (a single DSB can be visualized), permitting the use of extremely small samples. Measurements of γ-H2AX focal numbers can reveal subtle changes found in the radiation-induced tissue bystander response, low dose radiation exposure, and in cells with mutations in genomic stability maintenance pathways. In addition, marking DNA DSBs in a nucleus with γ-H2AX is a powerful tool to identify novel DNA repair proteins by their abilities to co-localize with γ-H2AX foci at the DSB site. This chapter presents techniques for γ-H2AX detection in a variety of human and mouse samples.