Analysis of copy number variations induced by ultrashort electron beam radiation in human leukocytes in vitro

Abstract

Background Environmental risk factors have been shown to alter DNA copy number variations (CNVs). Recently, CNVs have been described to arise after low-dose ionizing radiation in vitro and in vivo. Development of cost- and size-effective laser-driven electron accelerators (LDEAs), capable to deliver high energy beams in pico- or femtosecond durations requires examination of their biological effects. Here we studied in vitro impact of LDEAs radiation on known CNV hotspots in human peripheral blood lymphocytes on single cell level. Results Here CNVs in chromosomal regions 1p31.1, 7q11.22, 9q21.3, 10q21.1 and 16q23.1 earlier reported to be sensitive to ionizing radiation were analyzed using molecular cytogenetics. Irradiation of cells with 0.5, 1.5 and 3.0 Gy significantly increased signal intensities in all analyzed chromosomal regions compared to controls. The latter is suggested to be due to radiation-induced duplication or amplification of CNV stretches. As significantly lower gains in mean fluorescence intensities were observed only for chromosomal locus 1p31.1 (after irradiation with 3.0 Gy variant sensitivites of different loci to LDEA is suggested. Negative correlation was found between fluorescence intensities and chromosome size ( r = − 0.783, p < 0.001) in cells exposed to 3.0 Gy irradiation and between fluorescence intensities and gene density ( r = − 0.475, p < 0.05) in cells exposed to 0.5 Gy irradiation. Conclusions In this study we demonstrated that irradiation with laser-driven electron bunches can induce molecular-cytogenetically visible CNVs in human blood leukocytes in vitro. These CNVs occur most likely due to duplications or amplification and tend to inversely correlate with chromosome size and gene density. CNVs can last in cell population as stable chromosomal changes for several days after radiation exposure; therefore this endpoint can be used for characterization of genetic effects of accelerated electrons. These findings should be complemented with other studies and implementation of more sophisticated approaches for CNVs analysis.

DNA Copy Number Variations as Markers of Mutagenic Impact

“…The authors claim that gains and losses of cancer-related candidate genes and miRNAs observed in studied cohort have the potential to allow identification of radiation-associated breast cancer at the individual level [37]. Interestingly, gains in chromosomal region 7q11.22 were also observed in our study of radiation-induced CNVs in human leukocytes in vitro [25] indicating feasibility of CNVs in this locus for detection of radiation exposure in human cells.…”

“…We studied CNVs in chromosomal regions 1p31.1, 7q11.22, 9q21.3, 10q21.1 and 16q23.1 in cultured normal human blood leukocytes irradiated with laser-driven electron bunches using parental origin determination FISH (POD-FISH) [25]. Irradiation of cells significantly increased levels of duplication in all analyzed chromosomal regions earlier reported to be sensitive to ionizing radiation.…”

Low-Energy Laser-Driven Ultrashort Pulsed Electron Beam Irradiation-Induced Immune Response in Rats

“…The investigations on the biological effect/effectiveness of conventional, not laserdriven, accelerators have been conducted for several decades [12][13][14][15][16][17][18]. However, since ultrashort pulsed laser-driven electron accelerators are relatively new, data about their radiobiological effect is very limited in the literature, and the existing studies are mostly related to in vitro studies [19][20][21][22][23][24]. Thus, laser-driven UPEB irradiation has been shown to induce molecular-cytogenetically visible copy number variations (CNVs) [19], increase micronucleus frequency, and shorten telomere length in healthy human peripheral blood lymphocytes [18,25].…”

“…However, since ultrashort pulsed laser-driven electron accelerators are relatively new, data about their radiobiological effect is very limited in the literature, and the existing studies are mostly related to in vitro studies [19][20][21][22][23][24]. Thus, laser-driven UPEB irradiation has been shown to induce molecular-cytogenetically visible copy number variations (CNVs) [19], increase micronucleus frequency, and shorten telomere length in healthy human peripheral blood lymphocytes [18,25]. In the human ovarian cancer cell line OVCAR-3, it was shown to reduce the cell survival [25].…”

Doxorubicin-Induced Translocation of mtDNA into the Nuclear Genome of Human Lymphocytes Detected Using a Molecular-Cytogenetic Approach

“…Metaphase chromosomes were prepared as previously described [ 38 ]. Forty-eight hours after culture initiation, DOX was added at concentrations of 0.025, 0.035 and 0.05 µg/mL and incubated for 24 h. Colcemid (0.1 μg/mL final concentration; Merck, Germany) was added to the culture 1.5 h before harvesting and incubated at 37 °C to achieve metaphase block.…”