X-irradiation of cells on glass slides has a dose doubling impact

Abstract

Immunofluorescence detection of γH2AX foci is a widely used tool to quantify the induction and repair of DNA double-strand breaks induced by ionising radiation. We observed that Xirradiation of mammalian cells exposed on glass slides induced 2-fold higher foci numbers compared to irradiation with γ-rays. Here, we show that the excess γH2AX foci after Xirradiation are produced from secondary radiation particles generated from the irradiation of glass slides. Both 120 kV X-rays and 137 Cs γ-rays induce ~20 γH2AX foci per Gy in cells growing on thin (~2 µm) plastic foils immersed in water. The same yield is obtained following γ-irradiation of cells growing on glass slides. However, 120 kV X-rays produce ~40 γH2AX foci per Gy in cells growing on glass, two-fold greater than obtained using cells irradiated on plastic surfaces. The same increase in γH2AX foci number is obtained if the plastic foil on which the cells are grown is irradiated on a glass slide. Thus, the physical proximity to the glass material and not morphological differences of cells growing on different surfaces accounts for the excess γH2AX foci. The increase in foci number depends on the energy and is considerably smaller for 25 kV relative to 120 kV X-rays, a finding which can be explained by known physical properties of radiation. The kinetics for the loss of foci, which is taken to represent the rate of DSB repair, as well as the Artemis dependent repair fraction, was similar following X-or γ-irradiation, demonstrating that DSBs induced by this range of treatments are repaired in an identical manner.3

Inducible response required for repair of low-dose radiation damage in human fibroblasts

“…Here, we investigated a range of doses between 2.5 and 200 mGy and show that the level of initial γ-H2AX and pATM foci at 5 min postirradiation depends linearly on dose, with a yield of ≈20 foci per 1 Gy per cell, similar to the yield obtained in previous studies with much higher doses (28) (Fig. 1C and Fig.…”

“…Thus, foci induction is similar in various mouse tissues and increases linearly with dose from 10 mGy to 1 Gy. The induction yield of about 7 foci per cell per 1 Gy at 10 min post-IR is similar to that of our previous study (34) but substantially lower than the level of 15-20 foci per 1 Gy typically observed for human cells in culture (28). We have previously suggested that this difference may reflect that mouse cells have a 20% lower DNA content than human cells, that additional foci loss can occur during the time necessary to retrieve and fix the organs, and that foci scoring in a cross-section of cells embedded in a tissue sample will significantly underestimate the true foci numbers (34).…”

“…Irradiation was performed with an x-ray machine (PW2184; Philips) at 90 kV, 6 mA, and a dose rate of 70 mGy/min, determined with a dosimeter (PTW-SN4, type 7612; PTW) and chemical dosimetry. We have considered in the present study that cells irradiated on glass slides receive a dose that is higher, as determined by physical and chemical dosimetry (28).…”

Direct measurement of the 3-dimensional DNA lesion distribution induced by energetic charged particles in a mouse model tissue

“…In all subsequent experiments, we analyzed foci at 15 min post-IR. The maximum number of nine γH2AX foci per photoreceptor is substantially lower than the level of approximately 20 foci per human cell in culture (34). However, other mouse tissues, such as the brain and kidney, with cells of different nuclear architecture and size also exhibit only approximately 10.5 foci per cell, suggesting that the difference with human cells in culture is not a specific phenomenon of photoreceptors (35) (Fig.…”

The Limitations of the G1-S Checkpoint

“…X-irradiation was performed at 90 or 120 kV. The increase in dose for cells grown on glass coverslips relative to plastic surfaces was taken into account in the dose assessment (20).…”