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.

Materials and 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.

Conclusion: 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.

Keywords::

• Ionizing radiation
• peripheral blood mono-nuclear cells (PBMC)
• DNA damage response
• chromatin fiber conformational changes
• Dynamic Light Scattering

Acknowledgements

We thank all the volunteers, who have participated and donated blood samples for our study. We are thankful to Ms Prabhu J. A. and Mr Sangram Kamble for helping us in collecting the blood samples from BARC dispensary, Modular Laboratories, Trombay, Mumbai. We acknowledge Mr Prayag Amin for his excellent technical assistance, while acquiring the samples in Flow Cytometry experiments. We thank Dr (Mrs) Bhavani Shankar for the inputs during flow cytometry experiment. We also acknowledge Ms Vasumathy and Shri Manzoor Ali for helping us during confocal microscopy. We are thankful to Dr (Mrs) Shilpa Sawant for helping us during AFM experiments. We express our sincere thanks to Dr M. Seshadri, Former Head, Radiation Biology and Health Sciences Division for useful discussion during the experiments. We profusely thank Dr K. B. Sainis, former Director, Bio-medical Group, for critical evaluation and useful discussions during the preparation of the manuscript. We also thank Dr J. R. Bandekar, Head, Radiation Biology and Health Sciences Division, for his useful discussions.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.