Abstract
The cytokinesis-block micronucleus (CBMN) assay is a standardized method used for genotoxicity studies. Conventional whole blood cultures (WBC) are often used for this assay, although the assay can also be performed on isolated peripheral blood mononuclear cell (PBMC) cultures. However, the standardization of a protocol for the PBMC CBMN assay has not been investigated extensively. The aim of this study was to optimize a reliable CBMN assay protocol for fresh and cryopreserved peripheral blood mononuclear cells (PBMCS), and to compare micronuclei (MNi) results between WBC and PBMC cultures. The G0 CBMN assay was performed on whole blood, freshly isolated, and cryopreserved PBMCS from healthy human blood samples and five radiosensitive patient samples. Cells were exposed to 220 kV X-ray in vitro doses ranging from 0.5 to 2 Gy. The optimized PBMC CBMN assay showed adequate repeatability and small inter-individual variability. MNi values were significantly higher for WBC than for fresh PBMCS. Additionally, cryopreservation of PBMCS resulted in a significant increase of MNi values, while different cryopreservation times had no significant impact. In conclusion, our standardized CBMN assay on fresh and cryopreserved PBMCS can be used for genotoxicity studies, biological dosimetry, and radiosensitivity assessment.
Highlights
Chromosomal damage can be assessed using various cytogenetic assays
The cytokinesis-block micronucleus (CBMN) assay was performed on whole blood cultures (WBC), fresh peripheral blood mononuclear cell (PBMC) and cryopreserved (2 w and 25 w) PBMC samples (Figure 1A)
The mean MNi response of WBC was significantly higher compared to fresh peripheral blood mononuclear cells (PBMCS)
Summary
Chromosomal damage can be assessed using various cytogenetic assays. These assays are often used in environmental biomonitoring studies, for example studies evaluating the genotoxic impact of nanoparticles on chromosomal damage, and in occupational genotoxicity studies, such as the influence of anesthetic gases in medical workers or the effect of cadmium exposure on battery manufacture workers [1,2,3,4]. The measurement of chromosomal damage induced by ionizing radiation is frequently used for radiation protection (e.g., biological dosimetry), as well as for radiobiological research (e.g., in vitro chromosomal radiosensitivity studies and prediction, or the follow up of radiation side effects) [3,7,8]. Ionizing radiation affects cells directly and indirectly, and can lead to an increase of the frequency of biological phenomena such as cell death, malignant transformations and chromosomal aberrations [9,10,11]. Cytogenetic biological dosimetry measures radiation-induced chromosomal aberrations as a proxy to estimate a received dose [12,13]. Over the past few decades, the use of chromosomal damage biomarkers has proven to be of value for dose assessments, especially when physical dosimetry data is insufficiently available [13]
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