Simultaneous induction of dispersed and clustered DNA lesions compromises DNA damage response in human peripheral blood lymphocytes

Abstract

Due to its ability to induce DNA damage in a space and time controlled manner, ionising radiation is a unique tool for studying the mechanisms of DNA repair. The biological effectiveness of ionising radiation is related to the ionisation density which is defined by the linear energy transfer (LET). Alpha particles are characterised by high LET, while X-rays by low LET values. An interesting question is how cells react when exposed to a mixed beam of high and low LET radiation. In an earlier study carried out with human peripheral blood lymphocytes (PBL) we could demonstrate that alpha radiation X-rays interact in producing more chromosomal aberrations than expected based on additivity. The aim of the present investigation was to look at the mechanism of the interaction, especially with respect to the question if it is due to an augmented level of initial damage or impaired DNA repair. PBL were exposed to various doses of alpha particles, X-rays and mixed beams. DNA damage and the kinetics of damage repair was quantified by the alkaline comet assay. The levels of phosphorylated, key DNA damage response (DDR) proteins ATM, p53 and DNA-PK were measured by Western blotting and mRNA levels of 6 damage-responsive genes were measured by qPCR. Alpha particles and X-rays interact in inducing DNA damage above the level predicted by assuming additivity and that the repair of damage occurs with a delay. The activation levels of DDR proteins and mRNA levels of the studied genes were highest in cells exposed to mixed beams. The results substantiate the idea that exposure to mixed beams presents a challenge for the cellular DDR system.

Fig 1. Dose response relationships for initial DNA damage.

Net relative tail intensity: percent DNA in the tail minus control. Symbols represent mean results from 3 independent experiments. Error bars represent standard deviations. *: significant (p<0.05) difference to alpha particles (one way ANOVA). Symbols are nudged for transparency. Exemplary images of comets following exposure to 0 Gy (control) and 2 Gy of alpha particles, X-rays and mixed beam are shown to the side of the graph.

Fig 2. Envelopes of additivity (circles and lines) for different levels of relative tail intensity (RTI), calculated for initial damage.

Squares represent the observed RTI in cells exposed to both alpha particles and X-rays at dose levels indicated, respectively, on the X and Y axes. The isobolograms and the observed RTI were derived from fits to dose-response relationships.

Fig 3

A: Repair kinetics following a dose of 2 Gy. Symbols represent mean results from 3 independent experiments. Relative tail intensity (percent): RTI normalized to initial level of damage. Error bars represent standard deviations. Symbols are nudged for transparency. B: Distributions of relative tail intensities at various time points post exposure.

Fig 4. Levels of phosphorylated proteins DNA-PKcs, ATM and p53 1h and 3h post exposure.

The relative level was calculated as fold increase in relation to GADPH and the average of each protein intensity of all samples. Mean results from 3 independent experiments for 1h and 5 independent experiments for 3h. Error bars represent standard errors. Asterisks represent significant differences at the level of * < 0.05, **<0.01 and ***<0.001 (Students t-test).

Fig 5. mRNA levels of DNA damage-responsive genes in human PBL after 4h, 24h and 48h incubation following 2 Gy exposure with X-rays, alpha particles and mixed beams.

Error bars represent standard errors. Asterisks represent significant differences at the level of * < 0.05, **<0.01 and ***<0.001 (Students t-test).