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. 2018 Jan 4;13(1):e0190330.
doi: 10.1371/journal.pone.0190330. eCollection 2018.

The Effect of Well-Characterized, Very Low-Dose X-Ray Radiation on Fibroblasts

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Free PMC article

The Effect of Well-Characterized, Very Low-Dose X-Ray Radiation on Fibroblasts

Katelyn Truong et al. PLoS One. .
Free PMC article

Abstract

The purpose of this study is to determine the effects of low-dose radiation on fibroblast cells irradiated by spectrally and dosimetrically well-characterized soft x-rays. To achieve this, a new cell culture x-ray irradiation system was designed. This system generates characteristic fluorescent x-rays to irradiate the cell culture with x-rays of well-defined energies and doses. 3T3 fibroblast cells were cultured in cups with Mylar® surfaces and were irradiated for one hour with characteristic iron (Fe) K x-ray radiation at a dose rate of approximately 550 μGy/hr. Cell proliferation, total protein analysis, flow cytometry, and cell staining were performed on fibroblast cells to determine the various effects caused by the radiation. Irradiated cells demonstrated increased proliferation and protein production compared to control samples. Flow cytometry revealed that a higher percentage of irradiated cells were in the G0/G1 phase of the cell cycle compared to control counterparts, which is consistent with other low-dose studies. Cell staining results suggest that irradiated cells maintained normal cell functions after radiation exposure, as there were no qualitative differences between the images of the control and irradiated samples. The result of this study suggest that low-dose soft x-ray radiation might cause an initial pause, followed by a significant increase, in proliferation. An initial "pause" in cell proliferation could be a protective mechanism of the cells to minimize DNA damage caused by radiation exposure. The new cell irradiation system developed here allows for unprecedented control over the properties of the x-rays given to the cell cultures. This will allow for further studies on various cell types with known spectral distribution and carefully measured doses of radiation, which may help to elucidate the mechanisms behind varied cell responses to low-dose x-rays reported in the literature.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Mylar® and individual cup components that make up the cell culture system.
Mylar® (A) provided a surface that allowed cells to grow in the same manner as if grown on standard tissue culture plastic, while also being thin enough to allow x-rays to pass through without significant attenuation. The Mylar® was pressed between the inner (B) and outer (C) cup components with a ventilation cap (D) to allow proper air-flow.
Fig 2
Fig 2. X-ray irradiator setup.
The radiation source (A) targeted the cell culture by fluorescing off the metal plate (B) before irradiating the culture through the Mylar® from the base of the cup (C).
Fig 3
Fig 3. Iron (Fe) XRF irradiation spectrum.
Displaying photon count vs. photon energy [keV].
Fig 4
Fig 4. Calibration spectrum.
Photon counts vs. photon energy [keV] of the SDD detector exposed to characteristic XRF emissions from a multivitamin target material for 900 seconds.
Fig 5
Fig 5. Proliferation assay.
Total cell number increased over time in culture for both the irradiated and control groups. However, while cells in the irradiated group had initially lower cell numbers one day after treatment (* = p<0.05), they proliferated much more quickly in the days following.
Fig 6
Fig 6. Total protein content.
(A) BCA Analysis over 4 days after irradiation (p<0.05). (B) The normalized data shows the change in protein levels increased more in the irradiated group than in the control, following a similar trend to that of the Proliferation Data.
Fig 7
Fig 7. Cell cycle analysis using flow cytometry.
Cells were measured to determine the percent positive in which they resided in the G0/G1 phase, S phase, and G2/M phase of the cell cycle (* = p<0.05).
Fig 8
Fig 8. Cell staining.
After irradiation, control samples (A) and irradiated samples (B) were stained for collagen (red), nuclei (blue), and cells (green) at 10X magnification on Day One after irradiation. Irradiated cells (C) stained to show collagen production.

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Grant support

The funding for this study was provided from NSF CBET CAREER 1245609 and the Clemson University Creative Inquiry program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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