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

Abstract

BackgroundMicronucleus (MN) assay is a well standardized approach for evaluation of clastogenic/aneugenic effects of mutagens. Fluorescence in situ hybridization (FISH) is successfully used to characterize the chromosomal content of MN. However, the relationships between nuclear positioning, length, and gene density of individual chromosomes and their involvement in MN induced by different mutagens have not been clearly defined.ResultsChromosomal content of MN was characterized in human leukocytes treated with mitomycin C (MMC) and bleomycin (BLM) by FISH using centromeric (cep) and whole-chromosome painting (wcp) probes. Involvement of chromosomes 8, 15 and 20 in MMC-induced and chromosomes 1, 9 and 16 in BLM-induced MN was studied, and correlated with chromosome size, gene density and interphase position. The results obtained were analyzed together with previous own data on the frequencies of inclusion of chromosomes 3, 4, 6, 7, 9, 16, 17, 18, and X in MMC-induced MN. It could be shown that MMC- and BLM-induced MN could contain material derived from all chromosomes investigated. Involvement of whole chromosomes 8, 15 and 20 in MMC-induced MN negatively correlated with gene density; however, analysis together with earlier studied chromosomes did not confirm this correlation. Inclusion of chromosomes 8, 15 and 20 in MMC-induced MN does not depend on their size and interphase position; the same result was found for the twelve overall analyzed chromosomes. In BLM-treated cells significant correlation between frequencies of involvement of chromosomes 1, 9 and 16 in MN and their size was found.ConclusionsOur results clearly revealed that BLM differs from MMC with respect to the distribution of induced chromosome damage and MN formation. Thus, DNA-damaging agents with diverse mechanism of action induce qualitatively different MN with regard to their chromosomal composition. Also this study demonstrates the utility of combined sequential application of cep and wcp probes for efficient detection of MN chromosomal content in terms of centric and acentric fragments.

Non-Random Mis-Segregation of Human Chromosomes

“…However, the question of whether these or additional characteristics generate bias in mis-segregation rates has not been answered to date since high-throughput methods to analyse chromosome-specific aneuploidy are lacking. The standard approach to measure aneuploidy, manual scoring of chromosome number using Fluorescence In-Situ Hybridization (FISH) of centromere-targeted probes (Burrell et al, 2013;Fenech, 2007) is low-throughput and subject to significant artefacts (Faggioli et al, 2012;Knouse et al, 2014;Valind et al, 2013;van den Bos et al, 2016), limiting the resolution of previous efforts to examine biased mis-segregation (Brown et al, 1983;Evans and Wise, 2011;Fauth et al, 1998;Hovhannisyan et al, 2016;Spence et al, 2006;Torosantucci et al, 2009;Xi et al, 1997). New technologies such as next generation sequencing-based methods van den Bos et al, 2016) are still expensive and technically challenging (Bakker et al, 2015).…”

Estimating genotoxic effects of anticancer drugs using cytokinesis-block micronucleus assay on human lymphocytes

“…В 17 работах использовали нативные лимфоциты крови человека, обработанные противоопухолевыми препаратами in vitro. Данный метод в экспериментах был дополнен другими цитогенетическими и молекулярно-генетическими подходами, такими как анализ хромосомных аберраций метафазных хромосом [8], метод флюоресцентной in situ гибридизации (FISH) [9], homozygotization assay [10], метод учета ДНК-комет [11].…”

“…Неоднократно описывался генотоксический потенциал цитостатических антибиотиков типа митомицина С и блеомицина, в том числе с помощью учета МЯ [9,13,14,15]. Данные препараты характеризуются направленным действием на молекулу ДНК и участвуют в формировании разрывов её цепей [16], ингибируют встраивание тимидина в молекулу ДНК, характеризуются усилением оксидативного стресса и повреждением митохондрий.…”

“…Разработка подходов по изучению вовлечения специфических хромосом в образование микроядер представляет собой новое направление молекулярной цитогенетической диагностики веществ, рассматриваемых в качестве генотоксикантов. С применением FISH с центромерными (cep) и whole-chromosome painting (wcp) пробами была установлена вовлеченность 8, 15 и 20 хромосом в формирование микроядер под действием митомицина С и 1, 9, 20 хромосом -при действии блеомицина [9].…”

Of Simple and Complex Genome Rearrangements, Chromothripsis, Chromoanasynthesis, and Chromosome Chaos

“…This suggests that the mechanisms leading to formation of small supernumerary marker chromosomes and chromothripsis may be distinct. However, more elaborate studies of chromosome distributions in micronuclei produced by precisely defined molecular mechanisms are needed to support or to reject some of the proposed putative causes of chromothripsis [Pellestor et al, 2014;Hovhannisyan et al, 2016;Nazaryan-Petersen and Tommerup, 2016].…”