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Influence of iodine supply on the radiation-induced DNA-fragmentation
Journal of Environmental Radioactivity xxx (2016) 1e5
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Journal of Environmental Radioactivity journal homepage: www.elsevier.com/locate/jenvrad
Influence of iodine supply on the radiation-induced DNA-fragmentation F. Sudbrock a, *, A. Herrmann a, T. Fischer a, B. Zimmermanns a, W. Baus b, A. Drzezga a, €cker a K. Schoma a b
Department of Nuclear Medicine, University Hospital of Cologne, Cologne 50924, Germany Department of Radiation Oncology, University Hospital of Cologne, Cologne 50924, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 November 2015 Received in revised form 6 July 2016 Accepted 8 July 2016 Available online xxx
The protective effect of stable iodide against radiation on thyroid cells was investigated. One physiological effect of stable iodine is well-rooted: stable iodine leads to a reduced thyroid uptake of radioactive iodine. This work wants to focus on an intrinsic effect of stable iodine by which DNA-damage in cells is prevented. To investigate this intrinsic effect thyroid cells (FRTL-5) were externally irradiated by use of a linear accelerator (LINAC) applying energy doses of 0.01 Gye400 Gy and by incubation with various activity concentrations of 131I (0.1e50 MBq/ml for 24 h). We added stable iodine (NaI) to the cells prior to external irradiation and investigated the effect of the concentration of stable iodine (1, 5, 15 mg/ml). In order to clarify whether thyroid cells have a distinctive and iodine-dependent reaction to ionizing radiation, keratinocytes (HaCaT) without NIS were exposed in the same way. As indicators for the cellular reaction, the extent of DNA fragmentation was determined (Roche, Mannheim, Germany). Both cell types showed distinct ability for apoptosis as proven with camptothecin. The addition of “cold” iodine from 1 to 15 mg/ml without irradiation (“negative control”) did not change the response in both cell types. Plausibly, the radio-sensitivity of both cell types did increase markedly with increasing radiation dose but the radiation effect is diminished if iodine is added to the thyroid cells beforehand. The DNA-damage in thyroid cells after addition of cold iodine is reduced by a factor of 2e3. The skin cells did not show an significant change of radio-sensitivity depending on the presence of cold iodine. Elementary iodine possibly acts as a radical scavenger and thus markedly reduces the secondary radiation damage caused by the formation of cytotoxic radicals. This intrinsic radioprotective effect of iodine is seen only in cells with NIS. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Radioiodine Radiation oncology Environmental radioactivity Radiation sensitivity Dosimetry
1. Introduction Non-radioactive iodine and thyrostatic drugs have long been known to have a significant influence on the therapeutic outcome of radioidine therapies [Schom€ acker et al., 1996] and it is common knowledge that stable iodine (“cold iodine”) competes with radioactive isotopes of iodine (“hot iodine”). The uptake of radioiodine may be significantly reduced when the concentration of stable iodine in the thyroid is high [Ramsden et al., 1967]. But apart form this well-know fact (“competiton effect”) it remains unclear if the competition of cold and hot iodine is the only influence of cold
* Corresponding author. E-mail address: [email protected] (F. Sudbrock).
iodine on the radiation sensitivity of cells. The aim of this work is to demonstrate that the iodine supply has an additional effect on the radiosensitivity of thyroid cells as cold iodine furthermore prevents DNA damage. We therefore studied the DNA fragmentation in irradiated cells after addition of iodine in physiological concentrations (mmol/l). The uptake of iodine into thyroid cells is regulated by the NaIsymporter (NIS) and depends on the iodine concentration [Kogai and Brent, 2012]. The radioiodine uptake in cells is limited by the presence of stable iodine due to competition for the NIS (“competition effect”) and inhibition of NIS expression, transient block of organification, and inhibition of hormonal release. This limitation of radioiodine uptake is undesirable for radioiodine therapy [Min et al., 2001], as it reduces the energy dose deposited in the thyroid and hence lessens the therapeutic effect. Conversely, it has
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F. Sudbrock et al. / Journal of Environmental Radioactivity xxx (2016) 1e5
been demonstrated that sufficient iodine supply reduces the risk of radiation-induced thyroid cancer after accidental exposure to radioiodine [Shakhtarin et al., 2003, Gembicki et al., 1997, Reiners and Schneider, 2013, Schneider and Smith, 2012]. This could also be explained by the reduced uptake of radioiodine due to the competiton effect. It is unclear whether iodine has an influence on its own to the radiosensitivity of cells due to its chemical properties, e.g. as a radical scavenger [Zhang et al., 2006, Yao et al., 2012]. Ionizing radiation transfers energy to cells either directly via cellular components and molecules or indirectly via water-derived radicals and reactive oxygen species (ROS) produced by radiolysis of water molecules. The latter react with nearby molecules within microseconds, resulting in breakage of chemical bonds or oxidation. A decisive contribution to radiation-induced cell damage is made by DNA breaks. Since DNA consists of a pair of complementary double strands cleavage can occur in either one single strand or both. However, the latter are believed to be much more critical biologically. Dose-effect relationships have been well established for several decades, with various indicators being used to quantify the effect induced by the radiation [Streffer and Müller 1987]. The use of CDDT for evaluation of radiation-induced cell damage is based on the detection of DNA-fragments as an indicator for cellular reactions. In recent years this test has become a commonly used measure of radiation-induced DNA fragmentation [Fischer et al., 2012]. The method was originally developed for detection of apoptosis. The splitting of DNA into oligonucleotides is taken as an indicator of the apoptotic process performed by specific endonucleases at the end of the signal cascade. Internucleosomal sections of DNA are split into histone-bound mono- or oligonucleosomes with about 180 base pairs, which can then be detected in the cytoplasm by means of anti-histone and anti-DNA antibodies. By the CDDT it is not feasible to distinguish whether the DNA strand breaks are directly generated by radiation or is a consequence of apoptotic pathways initiated by irradiation. Thyroid cells were externally irradiated by use of a linear accelerator (LINAC) and after incubation with various concentrations of radioiodine (Na131I). For the different types of radiation exposure we investigated the influence of the concentration of stable iodine added to the cells prior to irradiation. In order to clarify that thyroid cells have a distinctive reaction to ionizing radiation, keratinocytes without NIS were exposed in the same way. The extent of DNA damage was determined as an indicator of the cellular reaction. 2. Materials and methods 2.1. Preparation of cells Thyroid cells of the Fischer rat thyroid cell line FRTL-5 show the typical behaviour of thyroid cells (TSH-dependent growth, NIS expression) and are therefore suitable for studying the iodine dependence of radiation sensitivity. FRTL-5 (provided by B. Meller, € ttingen, Germany) and human adult low calcium Halle and Go temperature keratinocytes (HaCaT) purchased from the German Cancer Research Centre (DKFZ, Heidelberg, Germany) were cultivated and prepared for irradiation in the way described by Fischer et al., 2012]. The number of cells was counted using a Fuchs-Rosenthal counting chamber (LO Laboroptik, Friedrichstal, Germany). 3*105 cells of both cell types were immersed in 10 ml culture medium. 100 l of this suspension was transferred into each well of a 96-well microtiter plate (Corning BV Life sciences, Amsterdam, The Netherlands). These plates were placed in an incubator under the conditions described (24 h). After a further 24 h 100 ml of medium was added. Each transfer was carried out in an isolated box.
A test of the CDDT was carried out with an alkaloid (camptothecin, CPT) which is known to induce apoptosis. 2.2. Irradiations External beams irradiations were performed with linear accelerators from a radiation therapy department of the University Hospital of Cologne. For organisational reasons two different LINACs were used for cell irradiations (Elekta SL 75-5, Elekta SLi). The Elekta SL 75-5 produces 5 MeV photon beams and the Elekta SLi (Elekta, Innsbruck, Austria) produces 6 MeV photon beams. The field dimensions were 38 38 or 36 36 cm2, respectively. The source-surface distance was 58.5 or 57.2 cm, respectively. 10 cm of polystyrene was used as back-scatter material. Isocentric irradiations were performed from different angles. Irradiations were performed using energy doses of 0.01, 0.11, 0.12, 1.0, 4.4, 5, 9.6, 12.5, 21, 25, 45.8, 50, 96, 100, 200 and 400 Gy. For irradiation with radioiodine in the chemical form of Na131I, the cells were incubated with four different radioactivity concentrations of 131I (0.1, 1, 10 and 50 MBq/ml 131I) for 24 h. 2.3. Detection of induced cellular reactions Nucleosomal DNA was assayed in the culture supernatant and in the cytoplasmic fraction by an enzyme-linked immunosorbent assay employing the Cell Death Detection ELISAplus kit (Cell death detection test CDDT, Roche, Mannheim, Germany) according to the manufacturer’s instructions. The photometric absorption (extinction) of samples and negative controls was determined using a SPECTRA classic reader (SLT, Salzburg, Austria) at 405 nm (reference wavelength 492 nm). The results are discussed as “enrichment factor”. This factor describes the ratio of absorption of irradiated cells and non-irradiated cells. The fundamental ability of both cell types to undergo apoptosis and the sensitivity of CDDT in detecting this were tested systematically by means of the Camptothecin-test (CPT-test). CPT is an alkaloid that causes single and double strand breaks in DNA by preventing the re-ligation of DNA and inducing apoptosis. CPT was added in the following concentrations: 0.1, 1, 2 and 4 mg/ml medium. 2.4. The intrinsic influence of “cold” iodine For each experiment (LINAC and 131I) a control group was irradiated after addition of “cold” anionic iodine form in the respective concentrations and incubated in the same way. The difference in absorption of irradiated and non-irradiated cells was calculated as described. 2.5. Dosimetry Different approaches were applied for the dose calculations for external and internal irradiations. In the case of incubation with Na131I dosimetry was based on the Monte Carlo N-Particle code (MCNP) (http://mcnp-green.lanl.gov/index.html) in the form of the EGS-ray code [Kleinschmidt, 2001]. Simulations were performed assuming an exposure time of 24 h and a cylindrical well geometry with a volume of 200 ml (height: 0.57 cm, radius: 0.335 cm). The cells were assumed to build a layer on the base of the cylinder (height: 0.001 cm, radius: 0.3 cm). Calculations were performed for the beta particles from I-131 in 10 keV intervals with a range from 0 to 810 keV. The release of g-rays was ignored because the specific absorbed fraction is low and g-rays therefore do not contribute significantly to the dose. 106 single decays emitting electrons according to the b-spectrum of 131I yielded individual tracks in the
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Both cell types showed after incubation with camptothecin DNA fragments (the ratio of absorption of irradiated cells and nonirradiated cells, the so-called enrichment factor > 1) and, therefore, the suitability of CDDT for detection of DNA fragments could be demonstrated. For 1 mg/ml CPT the enrichment factor rises by a factor of 2e3 and then remains roughly constant for higher concentrations of CPT. The addition of “cold” iodine alone from 1 to 15 mg/ml without irradiation (“negative control”) does not change the enrichment factors (ratio of absorption of irradiated cells and non-irradiated cells). After irradiation the enrichment factors indicative of apoptosislike intracellular DNA fragments and necrosis-like extracellular DNA fragments increase markedly in the majority of cases with increasing radiation dose. These enrichment factors range from 1 (no radiation effect) to 12 (strong effect). The radiation effect is reduced if cold iodine is added to the cell beforehand (Figs. 1e6). The onset of radiation induced effects is seen for doses below 20 Gy but can not distinctively be specified. The results for thyroid cells and for keratinocytes in relation to iodine concentrations are summarized below. For LINAC irradiations a maximum for the overall uncertainty of the energy dose of 3.4% can be assumed and for the incubation with a radionuclide the error can be estimated with 6%.
Ratio of absorption irradiated vs. non irradiated cells
3. Results
10
without iodide 1 μg/ml iodide 5 μg/ml iodide 15 μg/ml iodide 5
0 0.01
0.10
1.00
10.00
100.00
1,000.00
Radiation Dose [Gy] Fig. 2. Thyroid cells (FRTL-5): Dose-dependent intensification of necrosis. DNA fragmentation after irradiation in the LINAC with various concentrations of iodine in the cell medium.
Ratio of absorption irradiated vs. non irradiated cells
given well geometries. Energy deposition was calculated from this and an S-factor derived [Fischer et al., 2012].
3
15 without iodide 15 μg/ml iodide
10
5
0 0.1
3.1. A. External irradiations: irradiations using a linear accelerator
Ratio of absorption irradiated vs. non irradiated cells
In this case an intense dose-dependent increase of intracellular DNA fragmentation between 0 and 100 Gy (Fig. 1) can be observed. This increase appears to begin to display a plateau at 200 Gy (enrichment factors 10e12) and is highest for cells where no stable iodine was added. For low levels of iodine added to the cell medium (1 mg/ml) the increase shows a smaller gradient. For an energy dose of 100 Gy the enrichment factor remains below 3 but we find a further increase of the enrichment factor with doses up to 400 Gy when it also reaches values of 10. At 400 Gy the enrichment factors are similar for cells with small amounts of iodine added or none. When higher amounts of stable iodine were added to the cells (5 and 15 mg/ml) the enrichment factors show a significant reduction by a factor of 2e3 even for the highest radiation doses applied. Here
10.0
100.0
1,000.0
Radiation dose [Gy] Fig. 3. Keratinocytes (HaCaT): Dose-dependent intensification of apoptosis. DNA fragmentation after irradiation in the LINAC with various concentrations of iodine in the cell medium.
Ratio of absorption irradiated vs. non irradiated cells
1. LINAC Apoptosis 1: Apoptosis-like intracellular DNA fragments FRTL (Fig. 1)
1.0
10 without iodine 15 μg iodine
5
0 1
10
100
1 ,00 0
Radiation Dose [Gy] Fig. 4. Keratinocytes (HaCaT): Dose-dependent intensification of necrosis. DNA fragmentation after irradiation in the LINAC with various concentrations of iodine in the cell medium.
15 without iodide 1 μg/ml iodide
10
the enrichment factor only reaches a maximum value below 5. For thyroid cells an unequivocal finding to be noted is that rising iodine concentrations between 1 and 15 mg/ml medium lead to a decrease in the enrichment factor and hence the radiation sensitivity determined for one end-point (DNA fragments).
5 μg/ml iodide 15 μg/ml iodide
5
0 0.01
0.10
1.00 10.00 100.00 Radiation dose [Gy]
1,000.00
Fig. 1. Thyroid cells (FRTL-5): Dose-dependent intensification of apoptosis. DNA fragmentation after irradiation in the LINAC with various concentrations of iodine in the cell medium.
2. LINAC Necrosis 1: Necrosis-like extracellular DNA fragments FRTL (Fig. 2) For thyroid cells the presence of necrosis-like extracellular DNAfragments again shows a dose-dependent but only slight increase. It indicates only a loose dependence on the concentration of iodine
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F. Sudbrock et al. / Journal of Environmental Radioactivity xxx (2016) 1e5
simulation for the geometric characteristics of a microtiter plate The simulations for this geometry yielded a 131I-dose per decay of 7.79 10 11 Gy Bq 1 s 1. With this dose factor the radiochemical concentrations of 0.1, 1, 10 and 50 MBq/ml were calculated in order to achieve energy doses in a comparable range of 0.6, 6.5, 65 and 322 Gy. As expected, for LINAC irradiations the most pronounced effect was found for the highest amount of iodine added to the medium of cells. Irradiations with 131I were therefore carried out with no iodine and 15 mg/ml iodine, respectively.
Ratio of absorption irradiated vs. non irradiated cells
10
without iodide 15 μg/ml iodide 5
5.
0 0.1
1.0
10.0
100.0
1,000.0
Radiation dose [Gy] Fig. 5. Thyroid cells (FRTL-5): Dose-dependent intensification of apoptosis. DNA fragmentation after incubation with Na131Iwith various concentrations of iodine in the cell medium.
Ratio of absorption irradiated vs. non irradiated cells
10
without iodide 15 μg/ml iodide 5
0 0.1
1.0
10.0
100.0
1,000.0
131
I Apoptosis 1: Apoptosis-like intracellular DNA fragments in FRTL-5 (Fig. 5)
For irradiations of thyroid cells with 131I the dose-dependent induction of intracellular DNA fragmentation is similar to that found for external irradiations using an accelerator (Fig. 5) but the enrichment factors are smaller by a factor of approximately 2 (maximum enrichment factor ~ 6). Interestingly if no iodine is added to FRTL-5 cells the enrichment factors already display a steep increase for comparatively low doses. For irradiations with 131I the addition of iodine reduces intracellular DNA fragmentation significantly between 10 Gy and 400 Gy by a factor of approximately 1.5e2. The radiation effect for 131I- irradiation is less pronounced and accordingly the effect of additional iodine is smaller. The shapes of both curves also display a saturation effect indicating, as discussed, a smaller extent of intracellular DNA fragmentation. 6.
131
I Apoptosis 2: Apoptosis-like intracellular DNA fragments in HaCaT (Fig. 6)
Radiation dose [Gy] Fig. 6. Keratinocytes (HaCaT): Dose-dependent intensification of apoptosis. DNA fragmentation after incubation with Na131Iwith various concentrations of iodine in the cell medium.
in the medium. A significant alteration in enrichment factor between 0 and 15 mg/ml can be seen only for high doses. 3. LINAC Apoptosis 2: Apoptosis-like intracellular DNA fragments HaCaT (Fig. 3) For keratinocytes with no stable iodine added the dose-effect relationship resembles that found for the respective thyroid cells. A levelling out in the dose-effect curve is found in the region below 100 Gy and the enrichment factors are also comparable to those measured in thyroid cells (10e12). But even when the highest concentration of stable iodine is added to the medium of keratinocytes no significant reduction in intracellular DNA fragmentation can be observed. 4. LINAC Necrosis 2: Necrosis-like extracellular DNA fragments HaCaT (Fig. 4) The irradiation of keratinocytes by means of a linear accelerator show no significant dose-dependent effects in relation to necrosislike extracellular DNA fragments. The total enrichment factor of necrotic cells remains relatively low even at very high doses. Nearly all enrichment factors are around 1. Again, for keratinocytes no difference is found between cells with and without added iodine. 3.2. B. Irradiations using The
dosimetric
131
I in comparison to irradiation by LINAC
calculations
performed
by
Monte-Carlo
The radiation effects for skin cells (Fig. 6) are detectable only with the highest dose of 400 Gy and are 4-fold less than those resulting from LINAC irradiation. The addition of 15 mg/ml has no protective effect against radiation. With respect to necrosis-like effects, FRTL cells show no significant reaction when exposed to the beta-gamma emitter 131I (enrichment factor ~ 1). Consequently, no difference is conceivable for cells with or without additional iodine. This finding also holds for keratinocytes: HaCaT cells react neither with apoptosis-like intracellular DNA fragments nor necrosis-like extracellular DNA fragments. 4. Discussion The role of iodine in the context of cytotoxic effects of radiation on (thyroid) cells has not yet been investigated, especially not with a focus on the chemical properties of iodine. The aim of this work was to demonstrate that the iodine supply has an additional effect on the radiosensitivity of thyroid cells apart from the wellestablished fact of thyroid blockage by stable iodine which has been exhaustively studied up to now: Cold iodine reduces radiation-induced DNA damage as well. The dose-dependent effect seems to be more distinctive for apoptosis. The efficiency to detect DNA fragments with the CDDT was proven using camptothecin (CPT). The induction of apoptosis by addition of camptothecin (CPT) displays saturation for higher concentrations of CPT but the ratio of absorption of irradiated cells and non-irradiated cells, the so-called enrichment factors, are markedly lower than those found after irradiation. Taking a closer look at the data from this study, the dosedependent radiation effects appear to display saturation beyond 100 Gy in many cases. As discussed by Stiblar-Martincic et al. on the basis of the morphometry of FRTL-5 cells, this cell-type is capable of
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repairing radiation-induced effects for a dose of 16 Gy whereas a dose of 24 Gy leads to irreparable damage [Stiblar-Martincic et al., 2000]. This finding is in agreement with the data presented here where apoptosis-like intracellular DNA fragments and necrosis-like extracellular DNA fragments remain low for doses not exceeding 20 Gy too far. In order to reproduce physiological conditions, the experiments described here were carried out with iodine concentrations in the order of mg/ml. This implies that the sodium iodine symporter (NIS) of all cells will surely be saturated so that the radionuclide will not be incorporated in the cell. The radionuclide remains in the surrounding medium and acts comparable to an external irradiation. Due to the range of the beta particles of 131I it is only of minor importance whether the radioiodine is deposited in the cell or not. We found significant differences in irradiated cells for different concentrations of stable iodine added. Where no stable iodine was added, the enrichment factors describing the DNA fragmentation display a steep increase for doses in the region of 20e100 Gy but only a moderate further increase for doses beyond 100 Gy. Increasing amounts of stable iodine added to the cells resulted in a reduction of radiation-induced effects. This finding appears to illustrate a radioprotective effect of stable iodine. The effect of reduced radiosensitivity after addition of iodine is prominently observable for thyroid cells (mM concentrations of stable iodine) and not for keratinocytes. Iodine plays an important role in molecular mechanisms within thyroid cells which might explain why the protective effect is found only in these types of cells and not in keratinocytes. One could argue that iodide (I ) itself or its oxidized form I2, which is formed in thyroid cells via thyroidperoxidase (TPO), are able to inactivate radical species. Obviously, cellular mechanisms like this oxidative process take place in FRTL-5 cells as discussed in the exemplary by Kimura et al. and Grollman et al. [Kimura et al., 2001 and Grollman et al., 1986]. It has in general been postulated that iodide or iodine acts as a radical scavenger on the cellular level. Various authors have postulated an antioxidative effect, comparable to that of vitamin C, here. Iodine containing thyroid hormones can also act as radical scavengers. The results presented here support these assumptions. 5. Conclusion An effect of stable iodine is unquestionable in connection with radioiodine therapies. The uptake of radioactive iodine into the cells will be markedly reduced. But this competition effect is in our opinion not a genuine radioprotective effect, as it rather depends on the physiological similarity of “cold” and “hot” iodine. With our findings we now assume that stable iodine prevents DNA-damage itself and therefore acts as a genuine radioprotective agent in cells. This “intrinsic” effect is exclusively observed for thyroid cells and leads to a reduced DNA-damage by a factor of 2e3. This assumption also implies that cells sufficiently supplied with iodine will be more resistant to both external and internal irradiation. The competition of “hot” and “cold” iodine would only explain a protective effect after incorporation of radioactivity. But,
5
of course, the radioprotective effect also holds for any accidental incorporation of radioiodine where stable iodine would then act as a “radioprotector” in two directions: by reducing the uptake of radioactivity into the cells and by reducing the radiation damage caused by incorporated iodine. In case of accidental exposure to radioiodine it is recommended to administer potassium iodide to prevent thyroid uptake. This work justifies the assumption that potassium iodide moreover reduces DNA damage.
Conflicts of interest None.
References Fischer, T., Sudbrock, F., Pomplun, E., Kriehuber, R., Winkler, J., Matzkies, M., Dellweg, A., Dietlein, M., Arnhold, S., Royer, H.D., Schicha, H., Hescheler, J., Schom€ acker, K., 2012. Cellular response on Auger- and Beta-emitting nuclides: human embryonic stem cells (hESC) vs. keratinocytes. Int. J. Radiat. Biol. 88, 9619e9671. Gembicki, M., Stozharov, A.N., Arinchin, A.N., Moschik, K.V., Petrenko, S., Khmara, I.M., Baverstock, K.F., 1997. Iodine deficiency in Belarusian children as a possible factor stimulating the irradiation of the thyroid gland during the Chernobyl catastrophe. Environ. Health Perspect. 105 (Suppl. 6), 1487e1490. Grollman, E.F., Smolar, A., Ommaya, A., Tombaccini, D., Santisteban, P., 1986. Iodine suppression of iodide uptake in FRTL-5 thyroid cells. Endocrinology 118, 2477e2482. Kimura, T., Van Keymeulen, A., Golstein, J., Fusco, A., Dumont, J.E., Roger, P.P., 2001. Regulation of thyroid cell proliferation by TSH and other factors: a critical evaluation of in vitro models. Endocr. Rev. 22, 631e656. Kleinschmidt, C., 2001. EGS-ray, ein Programm zur Visualisierung von Monte-Carlo Rechnungen in der Strahlenphysik. Z Med. Phys. 11, 119e123 (in German). Kogai, T., Brent, G.A., 2012. The sodium iodide symporter (NIS): regulation and approaches to targeting for cancer therapeutics. Pharmacol. Ther. 135, 355e370. Min, J.J., Chung, J.K., Lee, Y.J., Jeong, J.M., Lee, D.S., Jang, J.J., Lee, M.C., Cho, B.Y., 2001. Relationship between expression of the sodium/iodide symporter and 131I uptake in recurrent lesions of differentiated thyroid carcinoma. Eur. J. Nucl. Med. 28, 639e645. Ramsden, D., Passant, F.H., Peabody, C.O., Speight, R.G., 1967. Radioiodine uptakes in the thyroid. Studies of the blocking and subsequent recovery of the gland followingthe administration of stable iodine. Health Phys. 633e646. Reiners, C., Schneider, R., 2013. Potassium iodide (KI) to block the thyroid from exposure to I-131: current questions and answers to be discussed. Radiat. Environ. Biophys. 52, 189e193. Schneider, A.B., Smith, J.M., 2012. Potassium iodide prophylaxis: what have we learned and questions raised by the accident at the Fukushima Daiichi Nuclear Power Plant. Thyroid 22, 344e346. €cker, K., Wellner, U., Scheidhauer, K., Gabruk-Szostak, B., Fischer, T., Schoma Steinbach, J., Füchtner, F., Schicha, H., 1996. Relationship between properties of 131 I therapy capsules and radioiodine kinetics. Nuklearmedizin 35, 175e180. Shakhtarin, V.V., Tsyb, A.F., Stepanenko, V.F., Orlov, M.Y., Kopecky, K.J., Davis, S., 2003. Iodine deficiency, radiation dose, and the risk of thyroid cancer among children and adolescents in the Bryansk region of Russia following the Chernobyl power station accident. Int. J. Epidemiol. 32, 584e591. Stiblar-Martincic, D., Pajer, Z., Cor, A., 2000. Morphometry of the FRTL-5 cells after irradiation. Acta Biol. Hung 51, 91e97. Streffer, C., Müller, W.U., 1987. Dose-effect relationships and general mechanisms of combined exposures. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 51, 961e969. Yao, Z., Li, M., He, J., Zhang, G., Wang, M., Ma, J., Sun, Y., Zhang, W., Li, L., 2012. Effect of early acute high concentrations of iodide exposure on mitochondrial superoxide production in FRTL cells. Free Rad. Biol. Med. 52, 1343e1352. Zhang, L., Sharma, S., Hershman, J.M., Brent, G.A., Dubinett, S.M., Huang, M., 2006. Iodide sensitizes genetically modified non-small cell lung cancer cells to ionizing radiation. Cancer Gene Ther. 13, 74e81.
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Journal of Environmental Radioactivity journal homepage: www.elsevier.com/locate/jenvrad
Influence of iodine supply on the radiation-induced DNA-fragmentation F. Sudbrock a, *, A. Herrmann a, T. Fischer a, B. Zimmermanns a, W. Baus b, A. Drzezga a, €cker a K. Schoma a b
Department of Nuclear Medicine, University Hospital of Cologne, Cologne 50924, Germany Department of Radiation Oncology, University Hospital of Cologne, Cologne 50924, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 November 2015 Received in revised form 6 July 2016 Accepted 8 July 2016 Available online xxx
The protective effect of stable iodide against radiation on thyroid cells was investigated. One physiological effect of stable iodine is well-rooted: stable iodine leads to a reduced thyroid uptake of radioactive iodine. This work wants to focus on an intrinsic effect of stable iodine by which DNA-damage in cells is prevented. To investigate this intrinsic effect thyroid cells (FRTL-5) were externally irradiated by use of a linear accelerator (LINAC) applying energy doses of 0.01 Gye400 Gy and by incubation with various activity concentrations of 131I (0.1e50 MBq/ml for 24 h). We added stable iodine (NaI) to the cells prior to external irradiation and investigated the effect of the concentration of stable iodine (1, 5, 15 mg/ml). In order to clarify whether thyroid cells have a distinctive and iodine-dependent reaction to ionizing radiation, keratinocytes (HaCaT) without NIS were exposed in the same way. As indicators for the cellular reaction, the extent of DNA fragmentation was determined (Roche, Mannheim, Germany). Both cell types showed distinct ability for apoptosis as proven with camptothecin. The addition of “cold” iodine from 1 to 15 mg/ml without irradiation (“negative control”) did not change the response in both cell types. Plausibly, the radio-sensitivity of both cell types did increase markedly with increasing radiation dose but the radiation effect is diminished if iodine is added to the thyroid cells beforehand. The DNA-damage in thyroid cells after addition of cold iodine is reduced by a factor of 2e3. The skin cells did not show an significant change of radio-sensitivity depending on the presence of cold iodine. Elementary iodine possibly acts as a radical scavenger and thus markedly reduces the secondary radiation damage caused by the formation of cytotoxic radicals. This intrinsic radioprotective effect of iodine is seen only in cells with NIS. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Radioiodine Radiation oncology Environmental radioactivity Radiation sensitivity Dosimetry
1. Introduction Non-radioactive iodine and thyrostatic drugs have long been known to have a significant influence on the therapeutic outcome of radioidine therapies [Schom€ acker et al., 1996] and it is common knowledge that stable iodine (“cold iodine”) competes with radioactive isotopes of iodine (“hot iodine”). The uptake of radioiodine may be significantly reduced when the concentration of stable iodine in the thyroid is high [Ramsden et al., 1967]. But apart form this well-know fact (“competiton effect”) it remains unclear if the competition of cold and hot iodine is the only influence of cold
* Corresponding author. E-mail address: [email protected] (F. Sudbrock).
iodine on the radiation sensitivity of cells. The aim of this work is to demonstrate that the iodine supply has an additional effect on the radiosensitivity of thyroid cells as cold iodine furthermore prevents DNA damage. We therefore studied the DNA fragmentation in irradiated cells after addition of iodine in physiological concentrations (mmol/l). The uptake of iodine into thyroid cells is regulated by the NaIsymporter (NIS) and depends on the iodine concentration [Kogai and Brent, 2012]. The radioiodine uptake in cells is limited by the presence of stable iodine due to competition for the NIS (“competition effect”) and inhibition of NIS expression, transient block of organification, and inhibition of hormonal release. This limitation of radioiodine uptake is undesirable for radioiodine therapy [Min et al., 2001], as it reduces the energy dose deposited in the thyroid and hence lessens the therapeutic effect. Conversely, it has
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been demonstrated that sufficient iodine supply reduces the risk of radiation-induced thyroid cancer after accidental exposure to radioiodine [Shakhtarin et al., 2003, Gembicki et al., 1997, Reiners and Schneider, 2013, Schneider and Smith, 2012]. This could also be explained by the reduced uptake of radioiodine due to the competiton effect. It is unclear whether iodine has an influence on its own to the radiosensitivity of cells due to its chemical properties, e.g. as a radical scavenger [Zhang et al., 2006, Yao et al., 2012]. Ionizing radiation transfers energy to cells either directly via cellular components and molecules or indirectly via water-derived radicals and reactive oxygen species (ROS) produced by radiolysis of water molecules. The latter react with nearby molecules within microseconds, resulting in breakage of chemical bonds or oxidation. A decisive contribution to radiation-induced cell damage is made by DNA breaks. Since DNA consists of a pair of complementary double strands cleavage can occur in either one single strand or both. However, the latter are believed to be much more critical biologically. Dose-effect relationships have been well established for several decades, with various indicators being used to quantify the effect induced by the radiation [Streffer and Müller 1987]. The use of CDDT for evaluation of radiation-induced cell damage is based on the detection of DNA-fragments as an indicator for cellular reactions. In recent years this test has become a commonly used measure of radiation-induced DNA fragmentation [Fischer et al., 2012]. The method was originally developed for detection of apoptosis. The splitting of DNA into oligonucleotides is taken as an indicator of the apoptotic process performed by specific endonucleases at the end of the signal cascade. Internucleosomal sections of DNA are split into histone-bound mono- or oligonucleosomes with about 180 base pairs, which can then be detected in the cytoplasm by means of anti-histone and anti-DNA antibodies. By the CDDT it is not feasible to distinguish whether the DNA strand breaks are directly generated by radiation or is a consequence of apoptotic pathways initiated by irradiation. Thyroid cells were externally irradiated by use of a linear accelerator (LINAC) and after incubation with various concentrations of radioiodine (Na131I). For the different types of radiation exposure we investigated the influence of the concentration of stable iodine added to the cells prior to irradiation. In order to clarify that thyroid cells have a distinctive reaction to ionizing radiation, keratinocytes without NIS were exposed in the same way. The extent of DNA damage was determined as an indicator of the cellular reaction. 2. Materials and methods 2.1. Preparation of cells Thyroid cells of the Fischer rat thyroid cell line FRTL-5 show the typical behaviour of thyroid cells (TSH-dependent growth, NIS expression) and are therefore suitable for studying the iodine dependence of radiation sensitivity. FRTL-5 (provided by B. Meller, € ttingen, Germany) and human adult low calcium Halle and Go temperature keratinocytes (HaCaT) purchased from the German Cancer Research Centre (DKFZ, Heidelberg, Germany) were cultivated and prepared for irradiation in the way described by Fischer et al., 2012]. The number of cells was counted using a Fuchs-Rosenthal counting chamber (LO Laboroptik, Friedrichstal, Germany). 3*105 cells of both cell types were immersed in 10 ml culture medium. 100 l of this suspension was transferred into each well of a 96-well microtiter plate (Corning BV Life sciences, Amsterdam, The Netherlands). These plates were placed in an incubator under the conditions described (24 h). After a further 24 h 100 ml of medium was added. Each transfer was carried out in an isolated box.
A test of the CDDT was carried out with an alkaloid (camptothecin, CPT) which is known to induce apoptosis. 2.2. Irradiations External beams irradiations were performed with linear accelerators from a radiation therapy department of the University Hospital of Cologne. For organisational reasons two different LINACs were used for cell irradiations (Elekta SL 75-5, Elekta SLi). The Elekta SL 75-5 produces 5 MeV photon beams and the Elekta SLi (Elekta, Innsbruck, Austria) produces 6 MeV photon beams. The field dimensions were 38 38 or 36 36 cm2, respectively. The source-surface distance was 58.5 or 57.2 cm, respectively. 10 cm of polystyrene was used as back-scatter material. Isocentric irradiations were performed from different angles. Irradiations were performed using energy doses of 0.01, 0.11, 0.12, 1.0, 4.4, 5, 9.6, 12.5, 21, 25, 45.8, 50, 96, 100, 200 and 400 Gy. For irradiation with radioiodine in the chemical form of Na131I, the cells were incubated with four different radioactivity concentrations of 131I (0.1, 1, 10 and 50 MBq/ml 131I) for 24 h. 2.3. Detection of induced cellular reactions Nucleosomal DNA was assayed in the culture supernatant and in the cytoplasmic fraction by an enzyme-linked immunosorbent assay employing the Cell Death Detection ELISAplus kit (Cell death detection test CDDT, Roche, Mannheim, Germany) according to the manufacturer’s instructions. The photometric absorption (extinction) of samples and negative controls was determined using a SPECTRA classic reader (SLT, Salzburg, Austria) at 405 nm (reference wavelength 492 nm). The results are discussed as “enrichment factor”. This factor describes the ratio of absorption of irradiated cells and non-irradiated cells. The fundamental ability of both cell types to undergo apoptosis and the sensitivity of CDDT in detecting this were tested systematically by means of the Camptothecin-test (CPT-test). CPT is an alkaloid that causes single and double strand breaks in DNA by preventing the re-ligation of DNA and inducing apoptosis. CPT was added in the following concentrations: 0.1, 1, 2 and 4 mg/ml medium. 2.4. The intrinsic influence of “cold” iodine For each experiment (LINAC and 131I) a control group was irradiated after addition of “cold” anionic iodine form in the respective concentrations and incubated in the same way. The difference in absorption of irradiated and non-irradiated cells was calculated as described. 2.5. Dosimetry Different approaches were applied for the dose calculations for external and internal irradiations. In the case of incubation with Na131I dosimetry was based on the Monte Carlo N-Particle code (MCNP) (http://mcnp-green.lanl.gov/index.html) in the form of the EGS-ray code [Kleinschmidt, 2001]. Simulations were performed assuming an exposure time of 24 h and a cylindrical well geometry with a volume of 200 ml (height: 0.57 cm, radius: 0.335 cm). The cells were assumed to build a layer on the base of the cylinder (height: 0.001 cm, radius: 0.3 cm). Calculations were performed for the beta particles from I-131 in 10 keV intervals with a range from 0 to 810 keV. The release of g-rays was ignored because the specific absorbed fraction is low and g-rays therefore do not contribute significantly to the dose. 106 single decays emitting electrons according to the b-spectrum of 131I yielded individual tracks in the
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Both cell types showed after incubation with camptothecin DNA fragments (the ratio of absorption of irradiated cells and nonirradiated cells, the so-called enrichment factor > 1) and, therefore, the suitability of CDDT for detection of DNA fragments could be demonstrated. For 1 mg/ml CPT the enrichment factor rises by a factor of 2e3 and then remains roughly constant for higher concentrations of CPT. The addition of “cold” iodine alone from 1 to 15 mg/ml without irradiation (“negative control”) does not change the enrichment factors (ratio of absorption of irradiated cells and non-irradiated cells). After irradiation the enrichment factors indicative of apoptosislike intracellular DNA fragments and necrosis-like extracellular DNA fragments increase markedly in the majority of cases with increasing radiation dose. These enrichment factors range from 1 (no radiation effect) to 12 (strong effect). The radiation effect is reduced if cold iodine is added to the cell beforehand (Figs. 1e6). The onset of radiation induced effects is seen for doses below 20 Gy but can not distinctively be specified. The results for thyroid cells and for keratinocytes in relation to iodine concentrations are summarized below. For LINAC irradiations a maximum for the overall uncertainty of the energy dose of 3.4% can be assumed and for the incubation with a radionuclide the error can be estimated with 6%.
Ratio of absorption irradiated vs. non irradiated cells
3. Results
10
without iodide 1 μg/ml iodide 5 μg/ml iodide 15 μg/ml iodide 5
0 0.01
0.10
1.00
10.00
100.00
1,000.00
Radiation Dose [Gy] Fig. 2. Thyroid cells (FRTL-5): Dose-dependent intensification of necrosis. DNA fragmentation after irradiation in the LINAC with various concentrations of iodine in the cell medium.
Ratio of absorption irradiated vs. non irradiated cells
given well geometries. Energy deposition was calculated from this and an S-factor derived [Fischer et al., 2012].
3
15 without iodide 15 μg/ml iodide
10
5
0 0.1
3.1. A. External irradiations: irradiations using a linear accelerator
Ratio of absorption irradiated vs. non irradiated cells
In this case an intense dose-dependent increase of intracellular DNA fragmentation between 0 and 100 Gy (Fig. 1) can be observed. This increase appears to begin to display a plateau at 200 Gy (enrichment factors 10e12) and is highest for cells where no stable iodine was added. For low levels of iodine added to the cell medium (1 mg/ml) the increase shows a smaller gradient. For an energy dose of 100 Gy the enrichment factor remains below 3 but we find a further increase of the enrichment factor with doses up to 400 Gy when it also reaches values of 10. At 400 Gy the enrichment factors are similar for cells with small amounts of iodine added or none. When higher amounts of stable iodine were added to the cells (5 and 15 mg/ml) the enrichment factors show a significant reduction by a factor of 2e3 even for the highest radiation doses applied. Here
10.0
100.0
1,000.0
Radiation dose [Gy] Fig. 3. Keratinocytes (HaCaT): Dose-dependent intensification of apoptosis. DNA fragmentation after irradiation in the LINAC with various concentrations of iodine in the cell medium.
Ratio of absorption irradiated vs. non irradiated cells
1. LINAC Apoptosis 1: Apoptosis-like intracellular DNA fragments FRTL (Fig. 1)
1.0
10 without iodine 15 μg iodine
5
0 1
10
100
1 ,00 0
Radiation Dose [Gy] Fig. 4. Keratinocytes (HaCaT): Dose-dependent intensification of necrosis. DNA fragmentation after irradiation in the LINAC with various concentrations of iodine in the cell medium.
15 without iodide 1 μg/ml iodide
10
the enrichment factor only reaches a maximum value below 5. For thyroid cells an unequivocal finding to be noted is that rising iodine concentrations between 1 and 15 mg/ml medium lead to a decrease in the enrichment factor and hence the radiation sensitivity determined for one end-point (DNA fragments).
5 μg/ml iodide 15 μg/ml iodide
5
0 0.01
0.10
1.00 10.00 100.00 Radiation dose [Gy]
1,000.00
Fig. 1. Thyroid cells (FRTL-5): Dose-dependent intensification of apoptosis. DNA fragmentation after irradiation in the LINAC with various concentrations of iodine in the cell medium.
2. LINAC Necrosis 1: Necrosis-like extracellular DNA fragments FRTL (Fig. 2) For thyroid cells the presence of necrosis-like extracellular DNAfragments again shows a dose-dependent but only slight increase. It indicates only a loose dependence on the concentration of iodine
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F. Sudbrock et al. / Journal of Environmental Radioactivity xxx (2016) 1e5
simulation for the geometric characteristics of a microtiter plate The simulations for this geometry yielded a 131I-dose per decay of 7.79 10 11 Gy Bq 1 s 1. With this dose factor the radiochemical concentrations of 0.1, 1, 10 and 50 MBq/ml were calculated in order to achieve energy doses in a comparable range of 0.6, 6.5, 65 and 322 Gy. As expected, for LINAC irradiations the most pronounced effect was found for the highest amount of iodine added to the medium of cells. Irradiations with 131I were therefore carried out with no iodine and 15 mg/ml iodine, respectively.
Ratio of absorption irradiated vs. non irradiated cells
10
without iodide 15 μg/ml iodide 5
5.
0 0.1
1.0
10.0
100.0
1,000.0
Radiation dose [Gy] Fig. 5. Thyroid cells (FRTL-5): Dose-dependent intensification of apoptosis. DNA fragmentation after incubation with Na131Iwith various concentrations of iodine in the cell medium.
Ratio of absorption irradiated vs. non irradiated cells
10
without iodide 15 μg/ml iodide 5
0 0.1
1.0
10.0
100.0
1,000.0
131
I Apoptosis 1: Apoptosis-like intracellular DNA fragments in FRTL-5 (Fig. 5)
For irradiations of thyroid cells with 131I the dose-dependent induction of intracellular DNA fragmentation is similar to that found for external irradiations using an accelerator (Fig. 5) but the enrichment factors are smaller by a factor of approximately 2 (maximum enrichment factor ~ 6). Interestingly if no iodine is added to FRTL-5 cells the enrichment factors already display a steep increase for comparatively low doses. For irradiations with 131I the addition of iodine reduces intracellular DNA fragmentation significantly between 10 Gy and 400 Gy by a factor of approximately 1.5e2. The radiation effect for 131I- irradiation is less pronounced and accordingly the effect of additional iodine is smaller. The shapes of both curves also display a saturation effect indicating, as discussed, a smaller extent of intracellular DNA fragmentation. 6.
131
I Apoptosis 2: Apoptosis-like intracellular DNA fragments in HaCaT (Fig. 6)
Radiation dose [Gy] Fig. 6. Keratinocytes (HaCaT): Dose-dependent intensification of apoptosis. DNA fragmentation after incubation with Na131Iwith various concentrations of iodine in the cell medium.
in the medium. A significant alteration in enrichment factor between 0 and 15 mg/ml can be seen only for high doses. 3. LINAC Apoptosis 2: Apoptosis-like intracellular DNA fragments HaCaT (Fig. 3) For keratinocytes with no stable iodine added the dose-effect relationship resembles that found for the respective thyroid cells. A levelling out in the dose-effect curve is found in the region below 100 Gy and the enrichment factors are also comparable to those measured in thyroid cells (10e12). But even when the highest concentration of stable iodine is added to the medium of keratinocytes no significant reduction in intracellular DNA fragmentation can be observed. 4. LINAC Necrosis 2: Necrosis-like extracellular DNA fragments HaCaT (Fig. 4) The irradiation of keratinocytes by means of a linear accelerator show no significant dose-dependent effects in relation to necrosislike extracellular DNA fragments. The total enrichment factor of necrotic cells remains relatively low even at very high doses. Nearly all enrichment factors are around 1. Again, for keratinocytes no difference is found between cells with and without added iodine. 3.2. B. Irradiations using The
dosimetric
131
I in comparison to irradiation by LINAC
calculations
performed
by
Monte-Carlo
The radiation effects for skin cells (Fig. 6) are detectable only with the highest dose of 400 Gy and are 4-fold less than those resulting from LINAC irradiation. The addition of 15 mg/ml has no protective effect against radiation. With respect to necrosis-like effects, FRTL cells show no significant reaction when exposed to the beta-gamma emitter 131I (enrichment factor ~ 1). Consequently, no difference is conceivable for cells with or without additional iodine. This finding also holds for keratinocytes: HaCaT cells react neither with apoptosis-like intracellular DNA fragments nor necrosis-like extracellular DNA fragments. 4. Discussion The role of iodine in the context of cytotoxic effects of radiation on (thyroid) cells has not yet been investigated, especially not with a focus on the chemical properties of iodine. The aim of this work was to demonstrate that the iodine supply has an additional effect on the radiosensitivity of thyroid cells apart from the wellestablished fact of thyroid blockage by stable iodine which has been exhaustively studied up to now: Cold iodine reduces radiation-induced DNA damage as well. The dose-dependent effect seems to be more distinctive for apoptosis. The efficiency to detect DNA fragments with the CDDT was proven using camptothecin (CPT). The induction of apoptosis by addition of camptothecin (CPT) displays saturation for higher concentrations of CPT but the ratio of absorption of irradiated cells and non-irradiated cells, the so-called enrichment factors, are markedly lower than those found after irradiation. Taking a closer look at the data from this study, the dosedependent radiation effects appear to display saturation beyond 100 Gy in many cases. As discussed by Stiblar-Martincic et al. on the basis of the morphometry of FRTL-5 cells, this cell-type is capable of
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repairing radiation-induced effects for a dose of 16 Gy whereas a dose of 24 Gy leads to irreparable damage [Stiblar-Martincic et al., 2000]. This finding is in agreement with the data presented here where apoptosis-like intracellular DNA fragments and necrosis-like extracellular DNA fragments remain low for doses not exceeding 20 Gy too far. In order to reproduce physiological conditions, the experiments described here were carried out with iodine concentrations in the order of mg/ml. This implies that the sodium iodine symporter (NIS) of all cells will surely be saturated so that the radionuclide will not be incorporated in the cell. The radionuclide remains in the surrounding medium and acts comparable to an external irradiation. Due to the range of the beta particles of 131I it is only of minor importance whether the radioiodine is deposited in the cell or not. We found significant differences in irradiated cells for different concentrations of stable iodine added. Where no stable iodine was added, the enrichment factors describing the DNA fragmentation display a steep increase for doses in the region of 20e100 Gy but only a moderate further increase for doses beyond 100 Gy. Increasing amounts of stable iodine added to the cells resulted in a reduction of radiation-induced effects. This finding appears to illustrate a radioprotective effect of stable iodine. The effect of reduced radiosensitivity after addition of iodine is prominently observable for thyroid cells (mM concentrations of stable iodine) and not for keratinocytes. Iodine plays an important role in molecular mechanisms within thyroid cells which might explain why the protective effect is found only in these types of cells and not in keratinocytes. One could argue that iodide (I ) itself or its oxidized form I2, which is formed in thyroid cells via thyroidperoxidase (TPO), are able to inactivate radical species. Obviously, cellular mechanisms like this oxidative process take place in FRTL-5 cells as discussed in the exemplary by Kimura et al. and Grollman et al. [Kimura et al., 2001 and Grollman et al., 1986]. It has in general been postulated that iodide or iodine acts as a radical scavenger on the cellular level. Various authors have postulated an antioxidative effect, comparable to that of vitamin C, here. Iodine containing thyroid hormones can also act as radical scavengers. The results presented here support these assumptions. 5. Conclusion An effect of stable iodine is unquestionable in connection with radioiodine therapies. The uptake of radioactive iodine into the cells will be markedly reduced. But this competition effect is in our opinion not a genuine radioprotective effect, as it rather depends on the physiological similarity of “cold” and “hot” iodine. With our findings we now assume that stable iodine prevents DNA-damage itself and therefore acts as a genuine radioprotective agent in cells. This “intrinsic” effect is exclusively observed for thyroid cells and leads to a reduced DNA-damage by a factor of 2e3. This assumption also implies that cells sufficiently supplied with iodine will be more resistant to both external and internal irradiation. The competition of “hot” and “cold” iodine would only explain a protective effect after incorporation of radioactivity. But,
5
of course, the radioprotective effect also holds for any accidental incorporation of radioiodine where stable iodine would then act as a “radioprotector” in two directions: by reducing the uptake of radioactivity into the cells and by reducing the radiation damage caused by incorporated iodine. In case of accidental exposure to radioiodine it is recommended to administer potassium iodide to prevent thyroid uptake. This work justifies the assumption that potassium iodide moreover reduces DNA damage.
Conflicts of interest None.
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Please cite this article in press as: Sudbrock, F., et al., Influence of iodine supply on the radiation-induced DNA-fragmentation, Journal of Environmental Radioactivity (2016), http://dx.doi.org/10.1016/j.jenvrad.2016.07.009