Assessment of individual radiosensitivity in human lymphocytes using micronucleus and microgel electrophoresis “Comet” assays

Background and purpose: Individual radiosensitivity is an inherent characteristic, associated with an increased reaction to ionizing radiation on the human body. Individuals show marked differences in radiation sensitivity, which has consequences in the fields of both radiation protection and radiation therapy. It is suggested that DNA repair mechanisms are involved. Consequently, the characterization of DNA repair in lymphocytes through cytokinesis blocked micronucleus (MN) and alkaline single-cell microgel electrophoresis (comet) assays could be suitable approaches to evaluate individual radiosensitivity in vitro. The aims of this study were: 1) to assess the in vitro radiosensitivity of peripheral blood lymphocytes from two groups of cancer patients (prospectively and retrospectively studied), using MN and comet assays, in comparison with the observed clinical response and 2) to test the predictive potential of both techniques. Materials and methods: 38 cancer patients receiving radiation therapy were enrolled in this study. The tumor sites were: head and neck (n = 25) and cervix (n = 13). 19 patients were evaluated prior, mid-way and on completion of treatment (prospective group) and 19 patients were evaluated about 2-480 month after radiotherapy (retrospective group). Cytogenetic data from the prospective group were analyzed using a mathematical model to evaluate the attenuation of the cytogenetic effect as a function of the time between a single exposure and blood sampling, estimating a cytogenetic recovery factor k. In the retrospective group, blood samples were irradiated in vitro with 0 (control) or 2 Gy and evaluated using MN test. Cytogenetic data were analyzed comparing expected MN frequencies (calibration curve from healthy donors) with values observed after in vitro irradiation. One over-reactor and patients that did not develop late effects were also evaluated through comet assay. DNA damage and repair capacity were quantified by the Olive tail moment. Lymphocytes of healthy individuals were used as reference sample. Results: In the prospective evaluation, factor k correlated with the individual radiosensitivity. Patients with low recovery from the cytogenetic effect (k tending to zero) developed late toxicity (fibrosis and actinic rectitis). In the retrospective evaluation, lymphocytes of 3 from 4 patients that had developed late reactions were significantly more radiosensitive than lymphocytes from the rest of the patients and normal donors. The individual cytogenetic response suggested a correlation with the maximum grade of late reaction (osteonecrosis, fibrosis and trismus). A patient with severe late reactions showed reduced DNA repair capacity, measured by comet assay. Conclusions: MN and comet tests could be suitable predictive assays to evaluate individual radiosensitivity in vitro. The identification of sub-groups with high radiosensitivity should be relevant for radiation protection and radiation therapy purposes. However, further studies are required to confirm the validity of these tests.