The Effect Of Calyculin A On The Dephosphorylation Of The Histone Gamma-H2ax After Formation Of X-Ray-Induced Dna Double-Strand Breaks In Human Blood Lymphocytes

Purpose : The purpose of this study was to investigate the effect of calyculin A on the number of gamma-H2AX foci (phosphorylated histone variant 2AX) in lymphocytes after in vitro and in vivo irradiation with rather low doses as they are used in diagnostic and interventional radiology.Materials and methods : For in vitro experiments blood samples of 14 healthy volunteers were irradiated with different doses (10, 50, 100 mGy) and incubated with (0.01, 0.1, 1, 10 nM) or without calyculin A for up to 2 hours. Non-irradiated samples with and without calyculin A served as controls. For in vivo evaluation blood samples were collected from seven patients undergoing computed tomography (CT) both with 1 nM calyculin A containing vials and vials without calyculin A. Foci were quantified in isolated lymphocytes using gamma-H2AX immunofluorescence microscopy.Results : 1 nM calyculin A led to a complete inhibition of gamma-H2AX foci loss in irradiated samples whereas no inhibition of p53 binding protein 1 (53 BP1) foci was found. Lower concentrations of the phosphatase inhibitor did not have a sufficient effect on foci decrease. Calyculin A did not affect foci levels in non-irradiated samples. If no calyculin A was added into the vial before the blood draws detectable CT-induced foci levels were lower in all patients with a reduction of the medians of 35%.Conclusions : Using gamma-H2AX immunofluorescence microscopy calyculin A can be a useful tool to mark the induced gamma-H2AX foci after low dose irradiation and to avoid an underestimation of the real deoxyribonucleic acid (DNA) damage in in vitro and in vivo experiments.