DNA Damage Focus Analysis in Blood Samples of Minipigs Reveals Acute Partial Body Irradiation

PLOS ONE, Dec 2019

Radiation accidents frequently involve acute high dose partial body irradiation leading to victims with radiation sickness and cutaneous radiation syndrome that implements radiation-induced cell death. Cells that are not lethally hit seek to repair ionizing radiation (IR) induced damage, albeit at the expense of an increased risk of mutation and tumor formation due to misrepair of IR-induced DNA double strand breaks (DSBs). The response to DNA damage includes phosphorylation of histone H2AX in the vicinity of DSBs, creating foci in the nucleus whose enumeration can serve as a radiation biodosimeter. Here, we investigated γH2AX and DNA repair foci in peripheral blood lymphocytes of Göttingen minipigs that experienced acute partial body irradiation (PBI) with 49 Gy (±6%) Co-60 γ-rays of the upper lumbar region. Blood samples taken 4, 24 and 168 hours post PBI were subjected to γ-H2AX, 53BP1 and MRE11 focus enumeration. Peripheral blood lymphocytes (PBL) of 49 Gy partial body irradiated minipigs were found to display 1–8 DNA damage foci/cell. These PBL values significantly deceed the high foci numbers observed in keratinocyte nuclei of the directly γ-irradiated minipig skin regions, indicating a limited resident time of PBL in the exposed tissue volume. Nonetheless, PBL samples obtained 4 h post IR in average contained 2.2% of cells displaying a pan-γH2AX signal, suggesting that these received a higher IR dose. Moreover, dispersion analysis indicated partial body irradiation for all 13 minipigs at 4 h post IR. While dose reconstruction using γH2AX DNA repair foci in lymphocytes after in vivo PBI represents a challenge, the DNA damage focus assay may serve as a rapid, first line indicator of radiation exposure. The occurrence of PBLs with pan-γH2AX staining and of cells with relatively high foci numbers that skew a Poisson distribution may be taken as indicator of acute high dose partial body irradiation, particularly when samples are available early after IR exposure.

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DNA Damage Focus Analysis in Blood Samples of Minipigs Reveals Acute Partial Body Irradiation

et al. (2014) DNA Damage Focus Analysis in Blood Samples of Minipigs Reveals Acute Partial Body Irradiation. PLoS ONE 9(2): e87458. doi:10.1371/journal.pone.0087458 DNA Damage Focus Analysis in Blood Samples of Minipigs Reveals Acute Partial Body Irradiation Andreas Lamkowski 0 1 Fabien Forcheron 0 1 Diane Agay 0 1 Emad A. Ahmed 0 1 Michel Drouet 0 1 Viktor Meineke 0 1 Harry Scherthan 0 1 Qinghua Shi, University of Science and Technology of China, China 0 Current address: Lab of Immunology and Molecular Physiology, Zoology Dept., Assiut University , Assiut , Egypt 1 1 Institut fu r Radiobiologie der Bundeswehr in Verb. mit der Universita t Ulm, M u nchen, Germany, 2 Institut de Recherche Biome dicale des Arme es (IRBA) , Bretigny sur Orge , France Radiation accidents frequently involve acute high dose partial body irradiation leading to victims with radiation sickness and cutaneous radiation syndrome that implements radiation-induced cell death. Cells that are not lethally hit seek to repair ionizing radiation (IR) induced damage, albeit at the expense of an increased risk of mutation and tumor formation due to misrepair of IR-induced DNA double strand breaks (DSBs). The response to DNA damage includes phosphorylation of histone H2AX in the vicinity of DSBs, creating foci in the nucleus whose enumeration can serve as a radiation biodosimeter. Here, we investigated cH2AX and DNA repair foci in peripheral blood lymphocytes of Go ttingen minipigs that experienced acute partial body irradiation (PBI) with 49 Gy (66%) Co-60 c-rays of the upper lumbar region. Blood samples taken 4, 24 and 168 hours post PBI were subjected to c-H2AX, 53BP1 and MRE11 focus enumeration. Peripheral blood lymphocytes (PBL) of 49 Gy partial body irradiated minipigs were found to display 1-8 DNA damage foci/cell. These PBL values significantly deceed the high foci numbers observed in keratinocyte nuclei of the directly c-irradiated minipig skin regions, indicating a limited resident time of PBL in the exposed tissue volume. Nonetheless, PBL samples obtained 4 h post IR in average contained 2.2% of cells displaying a pan-cH2AX signal, suggesting that these received a higher IR dose. Moreover, dispersion analysis indicated partial body irradiation for all 13 minipigs at 4 h post IR. While dose reconstruction using cH2AX DNA repair foci in lymphocytes after in vivo PBI represents a challenge, the DNA damage focus assay may serve as a rapid, first line indicator of radiation exposure. The occurrence of PBLs with pan-cH2AX staining and of cells with relatively high foci numbers that skew a Poisson distribution may be taken as indicator of acute high dose partial body irradiation, particularly when samples are available early after IR exposure. - The growing application of ionizing radiation (IR) in clinical therapeutic procedures and radiation accidents are major sources of human IR exposure. Irradiation accident patterns fall largely into the category of partial body irradiation (PBI) with an inhomogeneous field distribution, while homogeneous total body irradiation occurred only rarely in reported radiation accidents [2,3]. In terms of clinical treatment decisions, it is crucial to rapidly obtain an estimate of the doses and exposure scenario to predict the expected severity of radiation-induced damages. Biodosimetry tools can provide dose estimations that are needed to predict the clinical courses and to prepare for medical treatments and resources. However, these assays often require days until results are available [4,5]. Comparisons of the biological consequences of radiation exposure in different tissues on the same individual are scarce, even in animal models. Here we made use of the minipig large animal model to investigate the suitability of the cH2AX DNA damage focus assay to detect PBI. In the last decade, the Go ttingen minipig model was established as a clinical model for radiation injuries of the cutaneous [1,6,7], the hematopoietic radiation syndrome [8] and for biodosimetry [9]. One prominent feature of the exposure to IR is the formation of DNA double strand breaks (DSBs) that threaten a cells survival. DSB-elicited phosphorylation of histone H2AX occurs at serine 139 in H2AX molecules (c-H2AX) surrounding the DSBs, primarily by the ATM kinase [10]. cH2AX serves as a platform for the DNA damage response that signals DSB formation and directs DNA repair (e.g., [11,12]). Immunostaining of cH2AX [13] and other factors of the DNA damage response at focal sites surrounding DSB sites has led to the establishment of the cH2AX focus assay in peripheral blood cells [14,15]. The cH2AX focus assay has been shown to disclose IR exposures and provide information on DSB formation in various exposure scenarios (e.g., [1619]). Numerous in vitro studies highlighted the validity of the cH2AX assay for rapid detection of ionizing radiation exposure and absorbed doses in biological dosimetry [2022]. In vivo animal studies with murine [ (...truncated)


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Andreas Lamkowski, Fabien Forcheron, Diane Agay, Emad A. Ahmed, Michel Drouet, Viktor Meineke, Harry Scherthan. DNA Damage Focus Analysis in Blood Samples of Minipigs Reveals Acute Partial Body Irradiation, PLOS ONE, 2014, Volume 9, Issue 2, DOI: 10.1371/journal.pone.0087458