ABSTRACTS – ORAL PRESENTATIONS - AMCA, spol. s r.o.

P41. WHY CAN WE SEE DIFFERENT RADIOSENSITIVITY OF LYMPHOCYTE SUBSETS?
Lenka Zárybnická 1 , Zuzana Šinkorová 1 , Zuzana Kročová 2 , Jiřina Vávrová 1
1
Department of Radiobiology, Faculty of Military Health Sciences, University of Defence,
Hradec Králové, Czech Republic; zarybnicka@pmfhk.cz
2
Institute of Mollecular Pathology, Faculty of Military Health Sciences, University of
Defence, Hradec Králové, Czech Republic
Lymphocytes exposed to gamma-ray irradiation experience a degradation of their
macromolecules, especially DNA. Cells unsuccessful in DNA repairing process
induce apoptosis. Analysis of apoptotic peripheral blood mononuclear cells (PBMC)
by phosphatidyl serine detection (Annexin-V antibody) after in vitro irradiation is
dose dependent thus PMBC can be used as in vitro sensitive biodosimetric markers
(Šinkorová et al. 2011). Nevertheless, it is not possible to detect the apoptotic fraction
by Annexin-V antibody after in vivo irradiation (our unpublished results) due to the early
in vivo elimination of apoptotic cells from peripheral blood by active scavenging system
(Bogdandi et al. 2010), thus only the not influenced cells or repaired cells can be in vivo
studied. Many of studies confirmed different radiosenzitivity of individual lymphocyte
subsets where especially B lymphocytes are the most radiosensitive (Girinsky et al.
1991).
In this work we studied the radiosenzitivity of peripheral blood T-lymphocytes,
B-lymphocytes and natural killers (NK cells). We were looking for changes between
individual lymphocyte subsets after in vivo irradiation ( 60 Co, gamma source) focusing
on the very early phase of apoptosis when a decrease of mitochondrial membrane
potential ( D
ψ) occurs and furthermore on the period which precede the apoptosis, the
cell repair process. At the site where DNA is damaged the DNA double-strand breaks
(DSB) are produced and DNA damage repair factors are accumulated on chromatin.
One of the key processes activated within minutes after the DSB induction is the
phosphorylation of histone H2AX at serine 139 (γ-H2AX). γ-H2AX expression assessment
has been successfully exploited as in vivo biodosimetric marker (Rothkamm and Horn
2009). Mitochondrial membrane potential changes occuring within very early phases of
apoptosis can be analyzed by D
ψ-sensitive probe (JC1) which has been successfully used
within many apoptotic studies. We established new protocols combined intracellular
detection of γ-H2AX, respective D
ψ changes, with extracellular immunophenotyping
surface markers specific for rat or porcine NK cells (CD8, CD161), T-lymphocytes (CD3,
CD4, CD8) and B-lymphocytes (CD45RA) which were analyzed by flow cytometry
(CyAn ADP, Beckman Coulter). Using the animal experimental models (Wistar rats and
large white pigs) which were influenced by in vivo irradiation the changes of γ-H2AX
expression, respective D
ψ decrease, within lymphocyte subsets were analyzed. Studies
were approved by the Ethical Committee of the Faculty of Military Health Sciences in
Hradec Kralove and by the Ethical Committee of the Ministry of Defence of the Czech
Republic.
In our previous studies we confirmed that each lymphocyte subset within both animal
models (porcine, rat) exerts its own characteristic in vivo radiosenzitivity. In this study,
136 Analytical Cytometry VII