Applicability of flow cytometry γH2AX assay in population studies: suitability of fresh and frozen whole blood samples

Laffón, Blanca; Sánchez-Flores, María; Fernández-Bertólez, Natalia; Pásaro, Eduardo; Valdiglesias, Vanessa

doi:10.1007/s00204-021-03009-z

Cited by 4 publications

(2 citation statements)

References 42 publications

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“…Freeze thaw stability was studied for whole blood and purified lymphocyte samples because it is important in the clinical setting and for investigations of population exposure ( 59 ). Fresh whole blood was stored in an ice bath for extended periods of 10 h, and purified lymphocytes were stored in a −80°C freezer for long periods of 8 months.…”

Section: Discussionmentioning

confidence: 99%

An estimate assay for low-level exposure to ionizing radiation based on mass spectrometry quantification of γ-H2AX in human peripheral blood lymphocytes

Zhao¹,

Qu²,

Li³

et al. 2022

Front. Public Health

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Exposure to environmental ionizing radiation (IR) is ubiquitous, and large-dose exposure to IR is known to cause DNA damage and genotoxicity which is associated with an increased risk of cancer. Whether such detrimental effects are caused by exposure to low-dose IR is still debated. Therefore, rapid and early estimation of absorbed doses of IR in individuals, especially at low levels, using radiation response markers is a pivotal step for early triage during radiological incidents to provide adequate and timely clinical interventions. However, there is currently a crucial shortage of methods capable of determining the extent of low-dose IR exposure to human beings. The phosphorylation of histone H2AX on serine 139 (designated γ-H2AX), a classic biological dosimeter, can be used to evaluate the DNA damage response. We have developed an estimation assay for low-level exposure to IR based on the mass spectrometry quantification of γ-H2AX in blood. Human peripheral blood lymphocytes sensitive to low-dose IR, maintaining low temperature (4°C) and adding enzyme inhibitor are proven to be key steps, possibly insuring that a stable and marked γ-H2AX signal in blood cells exposed to low-dose IR could be detected. For the first time, DNA damage at low dose exposures to IR as low as 0.01 Gy were observed using the sensitive variation of γ-H2AX with high throughput mass spectrometry quantification in human peripheral blood, which is more accurate than the previously reported methods by virtue of isotope-dilution mass spectrometry, and can observe the time effect of DNA damage. These in vitro cellular dynamic monitoring experiments show that DNA damage occurred rapidly and then was repaired slowly over the passage of post-irradiation time even after exposure to very low IR doses. This assay was also used to assess different radiation exposures at the in vitro cellular level. These results demonstrate the potential utility of this assay in radiation biodosimetry and environmental risk assessment.

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Section: Discussionmentioning

confidence: 99%

An estimate assay for low-level exposure to ionizing radiation based on mass spectrometry quantification of γ-H2AX in human peripheral blood lymphocytes

Zhao¹,

Qu²,

Li³

et al. 2022

Front. Public Health

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“…Therefore, the evaluation of DSBs location and number may be used as a quantitative biomarker of genomic damage. Indeed, several γ-H2AX-based methods for the quantitative analysis of DSBs including uorescence microscopy [12,13] and ow cytometry [14,15] were successfully developed, also in the research on bleomycin-induced DSBs. For instance, γ-H2AX immunoimaging was applied by Liu and coworkers[16] to investigate the effect of four chemotherapeutic agents, including bleomycin on DNA damage in telomers in the C18-4 spermatogonial cell line.…”

Section: Introductionmentioning

confidence: 99%

Alterations in DNA conformation and histone phosphorylation resulting from double-strand breaks induced in HeLa cells – a spectroscopic and immunostaining approach

Czaja,

Giergiel,

Skirlińska-Nosek

et al. 2023

Preprint

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All living cells are subjected daily to several thousands of DNA lesions of various origins. DNA damage leads to genetic material disorders that may have serious consequences, including cell apoptosis or mitotic death. The most dangerous DNA lesions are double-strand breaks (DSBs) accompanied by modifications of the DNA chemical structure. Here, we focus on DNA conformational transitions caused by DSBs formation induced by a genotoxic drug, bleomycin (BLM). The response of cells to this anticancer agent treatment was monitored with synchrotron radiation infrared micro-spectroscopy (SR-FTIR) and confocal laser scanning microscopy (CLSM). Obtained data were analyzed using multivariate data analysis methods. Depending on BLM concentrations, either DNA repair or apoptosis was induced. The applied methodology enabled tracking of molecular modifications on those processes, such as conformational transition from B-DNA to A-DNA and increased expression of proteins within the cell nuclei resulting from the activation of repair processes. Finally, we present a novel approach to determine the rate of chromatin damage based on computational analysis of the histone H2AX phosphorylation immunoassay.

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Salivary leucocytes as suitable biomatrix for the comet assay in human biomonitoring studies

et al. 2021

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Peripheral blood leucocytes (PBL) have been traditionally used to investigate DNA damage by the comet assay in population studies, but validating alternative non-invasive samples would expand the application of this assay in human biomonitoring. The objectives of this study were (i) to test the validity of salivary leucocytes as a proper biomatrix for the comet assay, (ii) to evaluate the ability of this approach to detect different types of primary and oxidative DNA damage, and (iii) to determine whether frozen salivary leucocytes are still suitable for displaying those types of DNA damage. Fresh and frozen leucocytes isolated from saliva samples (six healthy non-smoking volunteers), were exposed to four genotoxic agents inducing different types of DNA damage, both primary (methyl methanesulfonate, actinomycin-D, ultraviolet radiation) and oxidative (potassium bromate), and standard or enzyme-modified comet assay was conducted. Results were compared with those obtained from PBL. Cells exposed to the four genotoxic agents showed dose-dependent increases of primary and oxidative DNA damage, demonstrating the suitability of all these samples to detect genetic damage from different origin. When comparing baseline levels of DNA damage, just a slight significant increase in primary DNA damage was observed in frozen salivary leucocytes regarding the other biomatrices, but similar results were obtained regarding sensitivity to DNA damage induction by all agents tested. This study demonstrates that salivary leucocytes can be employed in comet assay as an alternative or complement to blood samples. Frozen salivary leucocytes were proved to be a very convenient sample in large biomonitoring studies.

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Applicability of flow cytometry γH2AX assay in population studies: suitability of fresh and frozen whole blood samples

Cited by 4 publications

References 42 publications

An estimate assay for low-level exposure to ionizing radiation based on mass spectrometry quantification of γ-H2AX in human peripheral blood lymphocytes

An estimate assay for low-level exposure to ionizing radiation based on mass spectrometry quantification of γ-H2AX in human peripheral blood lymphocytes

Alterations in DNA conformation and histone phosphorylation resulting from double-strand breaks induced in HeLa cells – a spectroscopic and immunostaining approach

Salivary leucocytes as suitable biomatrix for the comet assay in human biomonitoring studies

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