Cytogenetically-based biodosimetry after high doses of radiation

Dosimetry is a crucial tool for triage and treatment planning following radiation exposure accidents. Biological dosimetry, which estimates exposure doses using biological parameters, offers a practical way to determine the specific dose an individual has received. The cytokinesis-blocked micronucleus (CBMN) assay is a well-established biodosimetric tool that measures chromosomal damage in mitogen-stimulated human lymphocytes. The CBMN method is particularly valuable for biodosimetry in triage situations due to its simplicity in scoring and adaptability to high-throughput automated sample processing systems. While this technique provides dose-response data that fits well with a linear-quadratic model for exposures to low linear energy transfer (LET) radiation and doses up to 5 Gy, its accuracy diminishes at higher doses. This limitation arises from the number of cells reaching mitosis. Although it is expected that micronuclei yield should increase with dose, many experiments have shown a decrease when normalized over the total number of cells, leading to variations that limit its accuracy for retrospective dose reconstruction.

In this study, we modified the standard CBMN assay to improve its accuracy for higher doses of photon radiation or mixed neutron-photon beams. The assay was modified by inhibiting the G2/M and spindle checkpoints with caffeine and/or ZM447439 (an Aurora kinase inhibitor), which were added to the blood cultures at specific times during the assay. Our results demonstrated that caffeine addition enhanced the assay performance for photon exposure up to 10 Gy. This improvement was achieved by extending the assay time from the typical 70 hours to 74 hours. When compared to micronuclei yields without inhibitors, the addition of caffeine and ZM447439 significantly improved the accuracy of detecting micronuclei yields up to 10 Gy for photons and 4 Gy for mixed neutrons-photons. Fitting dose-effect curves to account for the turnover phenomenon observed at higher doses revealed that the combination of both inhibitors provided the best fit. These modifications enable reliable dose reconstruction for high radiation doses and can be adapted to high-throughput automated ZM 447439 processing systems.