Radiation-induced conformational changes in chromatin structure in resting human peripheral blood mononuclear cells.
Abstract Background: Ionizing radiation induces a plethora of DNA damage including double-strand breaks (DSB) that may trigger a series of events such as transcription, DNA repair and alteration in the conformation of chromatin structure in human cells. We have made an attempt to study the conformational changes in chromatin fibers in irradiated human peripheral blood mononuclear cells (PBMC) using Dynamic Light Scattering (DLS) as a new tool.


METHODS
Venous blood samples were collected from 10 random, healthy individuals with written informed consent, approved by institutional ethics committee. PBMC were separated from blood, irradiated with different doses of gamma radiation from 0.25-1.0 Gy. Native chromatin was isolated from irradiated PBMC and changes in the hydrodynamic diameter of the chromatin fiber were measured using DLS. Both dose response and time kinetics was studied in order to see the chromatin changes. Radiation-induced DNA double-strand breaks were measured using gamma-H2AX (histone 2A member X) as a biomarker using flow cytometry and foci were visualized in confocal microscopy.


RESULTS
A significant alteration in hydrodynamic diameter of the chromatin fiber was observed at lower doses (0.25 and 0.50 Gy), whereas at higher dose (1.0 Gy), the size of the chromatin fiber was comparable to unirradiated control. Among the 10 individuals studied, five individuals showed significant increase (p ≤ 0.002) in hydrodynamic size at 0.25 Gy whereas four individuals showed significant decrease (p ≤ 0.009) at 0.25 Gy. One individual did not show any significant difference as compared to control. However, dose-dependent increase in gamma-H2AX fluorescence signals as well as foci number was observed. Increased fragmentation of chromatin fiber was also observed using Atomic Force Microscopy at higher doses.


CONCLUSIONS
Radiation-induced DNA damage response can lead to individual specific conformational changes in chromatin structure at lower doses (0.25 Gy and 0.50 Gy) which can be detected using dynamic light scattering method in resting human PBMC.
Low Level Radiation Research Section, Radiation Biology and Health Sciences Division, Bio Science Group, Bhabha Atomic Research Centre , Trombay, Mumbai-400 085 , India.
Vinay
V
Jain
P A
PA
Hassan
Birajalaxmi
B
Das
D002843:Chromatin / Q000737:chemistry* / Q000235:genetics; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D053938:DNA Fragmentation / Q000528:radiation effects; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000528:radiation effects*; D011487:Protein Conformation / Q000528:radiation effects
D002843:Chromatin
DNA damage response; Dynamic Light Scattering; Ionizing radiation; chromatin fiber conformational changes; peripheral blood mono-nuclear cells (PBMC)