Multicolour FISH analysis of ionising radiation induced micronucleus formation in human lymphocytes

Abstract

Micronucleation of chromosomal DNA is an effective indicator of DNA damage and micronucleus (MN) analysis is a valuable tool for radiation biodosimetry studies. To gain a comprehensive knowledge of micronucleation process after ionising radiation (IR) exposure, whole genome-wide chromosome analysis is desirable. With this objective, multicolour fluorescence in situ hybridization (M-FISH) technique was utilised in the present study to characterise the chromosome content of spontaneous and IR-induced micronuclei in three human donors. M-FISH analysis revealed a radiation dose-dependant increase in the number of micronuclei with multi-chromosome material above 2 Gy and as many as 3-6 multicolour signals were detected in micronuclei after high γ-rays radiation doses (5-10 Gy). Involvement of each human chromosome material was more frequently detected in multicoloured micronuclei than in single-coloured micronuclei at high radiation doses (>2 Gy). Observation of dose-dependant increase in the MN frequency with multi-chromosome material may be due to misrepair of DNA double-strand breaks involving multiple chromosomes leading to asymmetric dicentric or ring chromosomes and acentric fragments. Chromosomes belonging to groups A (1, 2 and 3) and B (4 and 5) were frequently detected in 35-45% of the total micronuclei either as single entities or in combination with other chromosomes. Among the A and B groups, chromosome 1 material was consistently detected at high MN frequencies after radiation exposure in all the donors. Additionally, chromosomes 13 and 19 were more frequently observed in micronuclei than the expected frequency based on DNA content. Our whole genome approach utilising the M-FISH technique revealed that MN formation at high radiation doses might be complex involving multiple chromosome fragments. Understanding the fate and biological consequences of these multi-chromosome-containing micronuclei may provide key molecular insights for some aspects of IR-induced genomic instability and cancer development processes.

Comparative analysis of individual chromosome involvement in micronuclei induced by mitomycin C and bleomycin in human leukocytes

“…The authors concluded that not all chromosomes in the human genome are equally susceptible to micronucleation. Balajee et al [ 12 ] confirmed results of Walker et al [ 11 ] on frequent involvement of larger chromosomes in radiation-induced MN. The authors consider that “frequencies of chromosomes micronucleation seem to correlate well with chromosome size because of a higher probability of double-strand breaks induction owing to spatial and temporal organization of chromatin in the interphase nuclei”.…”

“…Random involvement of chromosomes in radiation-induced MN, depending on the DNA content, was shown by [ 10 ]. Other studies demonstrated both random and non-random incorporation of DNA from different chromosomes in radiation-induced MN, larger chromosomes being usually overrepresented in the MN content [ 11 , 12 ].…”

Biomarkers of Ionizing Radiation Exposure: A Multiparametric Approach

“…A recent study utilized the multicolor FISH technique to identify the chromosomes involved in micronuclei formation after radiation exposure. [ 18 ] Recently, a new high-throughput and miniaturized CBMN method was also established for rapid processing a large number of samples (in about 3 days), a critical requirement for radiological triage. [ 19 20 ] Since the CBMN assay, unlike the DCA, can be easily performed and analyzed without an extensive cytogenetic expertise, many laboratories routinely use the CBMN assay for dose estimation.…”

MnSOD downregulation induced by extremely low 0.1 mGy single and fractionated X-rays and microgravity treatment in human neuroblastoma cell line, NB-1

“…The minimum dose is dependent on study endpoints, and the smaller the minimum dose, the more sensitive the endpoint. Studies of chromosomal aberration and micronucleus formation, ( 7 – 12 ) as well as mutation and transformation assays, ( 13 , 14 ) are sensitive approaches that utilize small doses of approximately 0.1 Gy (100 mGy). However, it remains controversial whether or not the dose-effect relationship is linear at lower doses, according to linear non-threshold (LNT), or non-LNT theory.…”