British Journal of Radiology (2005) 78, 335-337
© 2005 British Institute of Radiology
doi: 10.1259/bjr/24426084
Further evidence for biological effects resulting from ionizing radiation doses in the diagnostic X-ray range
S Anoopkumar-Dukie, MSc1,
A McMahon, BSc, HND2,
A Allshire, PhD1 and
T J Conere, MSc, PhD2
1 Department of Pharmacology and Therapeutics, University College Cork, Wilton, Cork, Ireland and 2 Medical Physics Department, Cork University Hospital, Wilton, Cork, Ireland
Correspondence: Dr Tom Conere, Medical Physics Department, Cork University Hospital, Wilton, Cork, Ireland
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Abstract
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In this study we assessed cell response to ionizing radiation doses in the diagnostic X-ray range, using metabolic performance as the biological end point. HeLa cells were irradiated between 3.5 mGy (standard deviation (SD=2.5%) and 55.1 mGy (SD=0.9%) with 110 kV X-rays, and after 7 days metabolic capacity of cultures was measured spectrophotometrically with resazurin. Cells receiving 7.5 mGy or greater showed impaired proliferation. At 7.5 mGy this effect was prevented by nitric oxide synthase inhibitor N G-monomethyl-L-arginine but not by ascorbic acid or p38 MAPK inhibitor SB203580. We conclude that HeLa cell proliferation is adversely affected by doses within the range of some radiological examinations, through a mechanism involving nitric oxide.
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Introduction
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There is currently much debate concerning the biological effects of low dose ionizing radiation [1–3] and whether a threshold dose exists for such effects. The International Commission on Radiological Protection (ICRP) [4] has adopted the so-called "linear-no threshold" hypothesis to estimate the risks for the stochastic effects of ionizing radiation. Under this hypothesis, the biological effect is assumed to be proportional to the radiation dose received, with no dose threshold for such effects. Hence the ICRP adopted a cautious approach in the light of scientific knowledge at that time and subsequently many national regulatory bodies have based their radiation exposure dose limits for occupationally exposed workers and members of the public on risk estimates derived using this model. However, there have been relatively few data obtained at low doses to support such an important concept. In this pilot study we have used enzymatic reduction of resazurin (Alamar Blue) to resorufin [5] as a sensitive indicator of cell response to the lower dose levels found in diagnostic radiology. We also assessed the effects of some potentially protective agents on radiation response.
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Methods
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The HeLa cell line purchased from American Type Culture Collection through LGC Promochem (Middlesex, UK) was routinely cultured in Minimal Essential Medium with Eagle's salts (Hepes modification) supplemented with 10% fetal bovine serum, gentamycin (20 µg ml–1), L-glutamine (2 mM), sodium bicarbonate (1.5 g l–1) and sodium pyruvate (1 mM). HeLa cells in exponential growth phase were irradiated to a range of doses from 3.5 mGy (standard deviation (SD)=2.5%) to 55.1 mGy (SD=0.9%) in 25 cm2 culture flasks, using an Argostat Plus TS-5 X-ray unit (Picker, Dublin, Ireland) operating at 110 kV. For irradiation, the flasks were inverted so that the surface to which the cells were attached was uppermost, to minimize dose variations due to meniscus effects etc. These acute radiation doses were delivered over 100 ms (3.5 mGy), 200 ms (7.5 mGy) and increasing to 250 ms for the higher dose points. Radiation output was measured prior to every exposure using a calibrated Unfors Mult-O-Meter radiation dose detector (Billdal, Sweden) inserted into a suitably modified culture flask similar to those used for irradiation of cells. These dose measurements were confirmed by thermoluminescent dosimetry, and total dose uncertainty was estimated at ±5%.
Following irradiation the cells were washed with phosphate buffered saline, trypsinised (0.25% w/v) and centrifuged at 400 x g. Upon resuspension in fresh culture medium, cells were counted using the trypan blue method and 5 x 103 trypan blue-excluding cells ml–1 were seeded in 96 well micro-titre plates. Culture medium over the cells was replaced at day 4 and again at day 7, in the latter case supplemented with resazurin (44 µM). Seven hours later resazurin reduction to resorufin was still within the linear range (<50%), and was measured spectrophotometrically at 570 nm and 600 nm using a Tecan Sunrise plate reader and X-read software (Salzburg, Austria).
Additional experiments were performed at 7.5 mGy, in which the antioxidant ascorbic acid (AA, 200 µM), nitric oxide synthase (NOS) inhibitor N G-monomethyl-L-arginine (LNMMA, 1 mM) or p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (10 µM) were added separately to cell cultures 40 min prior to irradiation, and remained until 10 min after irradiation, for a total period of 1 h. Data were analysed using t-test and Graphpad Instat software (California, USA). Significance levels were defined as p<0.05 (*), p<0.01 (**) and p<0.001 (***).
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Results
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Figure 1a
shows that irradiation over the range 7.5–55.1 mGy significantly impaired subsequent cell proliferation, as indicated by resazurin reduction capacity of cultures, whereas response of cells irradiated at 3.5 mGy was not distinguishable from controls. More detailed examination of the effect at 7.5 mGy (Figure 1b) showed that inhibition of NO production with the NOS inhibitor LNMMA abolished this irradiation effect. By contrast, presence of the antioxidant ascorbic acid during cell irradiation did not confer protection. Nor did the p38 MAPK inhibitor SB203580, which had been found in separate experiments to protect HeLa cells irradiated at 5 Gy.
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Figure 1. (a) Response of HeLa cells to low-dose X-ray irradiation, measured 7 days post irradiation as capacity for resazurin reduction. Data are means and standard deviation (SD) for three independent experiments, except for control (zero dose) and 7.5 mGy where n=6. (b) Protection against the irradiation effect at 7.5 mGy by LNMMA (1 mM) but not by ascorbic acid (AA, 200 µM) or p38 MAPK inhibitor SB203580 (10 µM). Data are means and (SD) for three independent experiments.
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Discussion
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The results presented in Figure 1
demonstrate adverse effects on HeLa cells of doses as low as 7.5 mGy, consistent with observations in other cell types and using different endpoints [2, 6]. Furthermore this present work implicates NO as a mediator of the effect at 7.5 mGy, on the basis of the protection afforded cells by the NOS inhibitor LNMMA. NO modulates cell radiosensitivity [7], and ionizing radiation increases NO production by inducing NOS expression [8] and stimulating constitutive NOS [9]. The cytotoxic effects of ionizing radiation (at higher doses than used here) have been attributed to mitochondria-dependent generation of both reactive oxygen and nitrogen species via an amplification mechanism involving mitochondrial permeability transition [10]. Other authors have described inhibition of mitochondrial respiratory complex I by NO [11] and the role of calcium signalling in activating NOS [12]. Thus changes in cellular calcium concentrations following radiation insult could lead to metabolic inhibition via increased production of NO. At present we are investigating these mechanisms further.
Since ascorbic acid is a good scavenger of both hydroxyl and peroxynitrite radicals, its failure to attenuate the effect of cell irradiation at 7.5 mGy suggests that neither species plays a critical role. Furthermore, at this low dose p38 MAPK is not implicated on the basis that p38 inhibitor SB203580 conferred no protection. By contrast, p38 inhibition fully protects HeLa cells irradiated at 5 Gy against metabolic impairment (unpublished observations).
The highly significant (p<0.001) 9.2% decrease in resazurin reduction at 7.5 mGy would imply a corresponding decrease of 4.3% at 3.5 mGy if a "linear-no-threshold" model applied. However we observed that resazurin reduction by cells irradiated at 3.5 mGy was marginally though not significantly stimulated. Thus these data leave open for now the question regarding the existence of a dose threshold.
In conclusion, this pilot study provides evidence of adverse effects to HeLa cells at 7.5 mGy, as measured by an assay of cellular proliferation. NO is a critical mediator of this injury but hydroxyl radical or peroxynitrite are probably not. Although we could not establish the existence of a dose threshold, if it exists then it must be below 7.5 mGy. In this context, other authors have reported cellular damage at 1.2 mGy [6]. Therefore further work is required to elucidate this point, given its importance for radiation protection and the whole concept of risk estimates based on the "linear no-threshold" model of radiation effects.
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Acknowledgments
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We acknowledge the assistance of Eugene O'Sullivan, Senior Physicist, Department of Medical Physics, Cork University Hospital and the financial support of the Cork based charity Aid Cancer Treatment.
Received for publication July 19, 2004.
Revision received September 24, 2004.
Accepted for publication December 7, 2004.
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References
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Abstract
Introduction
