In vitro micronucleus-centromere assay to detect radiation-damage induced by low doses in human lymphocytes

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Abstract
One of the major drawbacks of the in vitro micronucleus (MN) assay for human lymphocytes is its reduced sensitivity for the detection of damage induced by low radiation doses, due to the high variability among the spontaneous MN frequencies. In this paper we investigated the enhancement of the sensitivity of the MN assay by analysing spontaneous and radiation-induced MN for the presence of centromeres. For this, in situ hybridization (FISH) with the human pancentromeric DNA probe, p82H, was performed. Our results revealed that a high percentage (73%) of the spontaneous MN contain a centromere. These centromere-positive MN indicate the presence of a whole chromosome/chromatid. After in vitro irradiation with low doses (0.1-2 Gy) 60Co gamma-rays mainly centromere-negative MN were induced while only a very small number of additional centromere-positive MN were formed. This demonstrates that radiation-induced MN mainly contain acentric fragments pointing to the clastogenic action of ionizing radiation. Furthermore, our data show that the sensitivity of the MN assay for low dose detection is increased by scoring only centromere-negative MN.

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