Maria Wendisch

Fully automated interpretation of ionizing radiation-induced γH2AX foci by the novel pattern recognition system AKLIDES®

Purpose: Assessment of phosphorylated histone H2AX (γH2AX) foci as a measure for double-strand breaks (DSB) is a common technique. Since visual interpretation is time-consuming and influenced by subjective factors, we adapted the pattern recognition algorithms of autoantibodies to automated reading of γH2AX foci. Materials and methods: DSB formation was assessed by detection of γH2AX foci after exposition of thyreocyte rat cell line to 188Re. We used pattern recognition algorithms of the automated fluorescence interpretation system AKLIDES® for evaluation of γH2AX foci. Manual investigation was performed by three laboratories involving five observers. The results were compared by determining correlation and inter-laboratory variability. Results: The study confirmed the adaptation of automated interpretation system AKLIDES® to automated assessment of γH2AX foci in irradiated cells. Both manual and automated quantification resulted in increasing focus numbers depending on dose. Comparison of automated reading with visual assessment for five manual observers resulted in a determination coefficient of R2 = 0.889. The inter-laboratory variability for five manual investigators of three laboratories was 38.4 %. Conclusion: The interpretation system AKLIDES® demonstrated a high correlation with visually observed results. High inter-laboratory variability found for manual investigations revealed the usefulness for a standardized technique for evaluation of γH2AX foci.

Reduction in clonogenic survival of sodium-iodide symporter (NIS)-positive cells following intracellular uptake of 99mTc versus 188Re

Abstract Purpose: Cellular radionuclide uptake increases the heterogeneity of absorbed dose to biological structures. Dose increase depends on uptake yield and emission characteristics of radioisotopes. We used an in vitro model to compare the impact of cellular uptake of 188Re-perrhenate and 99mTc-pertechnetate on cellular survival. Materials and methods: Rat thyroid PC Cl3 cells in culture were incubated with 188Re or 99mTc in the presence or absence of perchlorate for 1 hour. Clonogenic cell survival was measured by colony formation. In addition, intracellular radionuclide uptake was quantified. Results: Dose effect curves were established for 188Re and 99mTc for various extra- and intracellular distributions of the radioactivity. In the presence of perchlorate, no uptake of radionuclides was detected and 188Re reduced cell survival more efficiently than 99mTc. A37, the activity that is necessary to yield 37% cell survival was 14 MBq/ml for 188Re and 480 MBq/ml for 99mTc. In the absence of perchlorate, both radionuclides showed similar uptakes; however, A37 was reduced by 30% for the beta-emitter and by 95% for 99mTc. The dose D37 that yields 37% cell survival was between 2.3 and 2.8 Gy for both radionuclides. Conclusions: Uptake of 188Re and 99mTc decreased cell survival. Intracellular 99mTc yielded a dose increase that was higher compared to 188Re due to emitted Auger and internal conversion-electrons. Up to 5 Gy there was no difference in radiotoxicity of 188Re and 99mTc. At doses higher than 5 Gy intracellular 99mTc became less radiotoxic than 188Re, probably due to a non-uniform lognormal radionuclide uptake.

[99mTc reduces clonogenic survival after intracellular uptake in NIS-positive cells in vitro more than 131I].

AIM In addition to gamma radiation of 140 keV 99mTc emits during the transition to 99Tc electrons of low energy and tiny path-lengths. These Auger electrons cannot be utilized in diagnostic procedures. However, they were discussed frequently for therapeutic application. Hitherto proof of effect of the Auger electrons from 99mTc is missing which is supplied now in an in vitro-system in comparison to beta-emitter 131I. METHODS The thyroid cell line PCCl3 (sodium iodide symporter (NIS)-positive) was incubated with 131I-sodium iodide (131I) or 99mTc-pertechnetate (99mTc) in presence or absence of perchlorate. For comparison the amount of radioactivity was adjusted to obtain the same dose from extracellular irradiation for both radionuclides. The colony forming assay detects the clonogenic cell survival as surviving fraction. In addition, intracellular radionuclide uptake was quantified. RESULTS Dose effect curves were established for 131I and 99mTc for variable extra- and intracellular distribution of the radioactivity. In presence of perchlorate no cellular uptake of radioactivity was detectable. Survival curves were largely comparable confirming the dosimetric calculations. In absence of perchlorate cellular radiotracer uptake varied from 1.39% (131I) to 1.90% 99mTc). Effects on survival were twice for the beta-emitter and ten-fold higher for 99mTc. CONCLUSIONS Intracellular uptake of 131I and 99mTc increases DNA-damage compared to strict extracellular radiotracer distribution which was demonstrated by means of colony forming assay. Increasing radiotoxicity from intracellular 99mTc is explained most likely by increased dose deposition in cellular structures due to Auger- and conversion-electrons of low range and high local energy deposition.

Cellular damage in vitro

AIM The cellular damage of ionising radiation depends on dose, physical radiation quality (e. g. LET) and intracellular radionuclide uptake. The influence of two beta emitters (188Re and 131I) on the thyroid cell line PCCl3 was studied. Furthermore, we analysed the effect of intracellular accumulation. METHODS The thyroid cell line PCCl3 was irradiated with 188Re-perrhenate or 131I-sodium iodide in presence or absence of perchlorate. The initial DNA-damage was measured in the comet assay as olive tail moment (OTM). The colony forming assay detects the clonogenic cell survival as surviving fraction. Additional the intracellular radionuclide uptake was quantified. RESULTS Dose response curves were established for irradiation with 188Re-perrhenate or 131I-iodine under various extra- and intracellular activity distribution conditions. In the presence of perchlorate DNA-damage and clonogenic cell survival for both radionuclides were comparable. In the absence of perchlorate radionuclide uptake of 1.39% (131I) and 4.14% (188Re) were measured causing twofold higher radiotoxicity. Although 131I uptake was lower than 188Re uptake the OTM values were higher und surviving fractions were lower. CONCLUSIONS 131I, compared to 188Re, has lower mean beta energy and a higher LET, and therefore, it induced a higher DNA-damage even at lower intracellular uptake. An additional explanation for the higher radiotoxicity of 131I could be the higher dose exposition caused by cross-fire through neighborhood cells.

Preincubation with Sn-complexes causes intensive intracellular retention of 99mTc in thyroid cells in vitro

UNLABELLED Technetium radiopharmaceuticals are well established in nuclear medicine. Besides its well-known gamma radiation, (99m)Tc emits an average of five Auger and internal conversion electrons per decay. The biological toxicity of these low-energy, high-LET (linear energy transfer) emissions is a controversial subject. One aim of this study was to estimate in a cell model how much (99m)Tc can be present in exposed cells and which radiobiological effects could be estimated in (99m)Tc-overloaded cells. METHODS Sodium iodine symporter (NIS)-positive thyroid cells were used. (99m)Tc-uptake studies were performed after preincubation with a non-radioactive (cold) stannous pyrophosphate kit solution or as a standard (99m)Tc pyrophosphate kit preparation or with pure pertechnetate solution. Survival curves were analyzed from colony-forming assays. RESULTS Preincubation with stannous complexes causes irreversible intracellular radioactivity retention of (99m)Tc and is followed by further pertechnetate influx to an unexpectedly high (99m)Tc level. The uptake of (99m)Tc pertechnetate in NIS-positive cells can be modified using stannous pyrophosphate from 3-5% to >80%. The maximum possible cellular uptake of (99m)Tc was 90Bq/cell. Compared with nearly pure extracellular irradiation from routine (99m)Tc complexes, cell survival was reduced by 3-4 orders of magnitude after preincubation with stannous pyrophosphate. CONCLUSIONS Intracellular (99m)Tc retention is related to reduced survival, which is most likely mediated by the emission of low-energy electrons. Our findings show that the described experiments constitute a simple and useful in vitro model for radiobiological investigations in a cell model.

DNA damage in lymphocytes after irradiation with 211At and 188Re

AIM Ionising radiation produces many types of DNA lesions of different complexity. High linear energy transfer (LET) types of radiation are biological more effective than low LET radiation. In the present work we applied the single cell gel electrophoreses (comet assay) to study the induction of initial DNA damage, efficiency of repair and residual DNA damage in lymphocytes after treatment with 211At and 188Re. METHODS Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood of healthy donors and irradiated with 211At and 188Re at different doses. The comet assay was performed under alkaline and neutral conditions in order to detect the initial DNA damage and its repair. The measure of damage was % tail DNA (percentage of DNA in the tail). RESULTS After treatment of cells with 188Re the initial DNA damage (% tail DNA) detected with the alkaline comet assay was higher than the damage measured for 211At. The neutral comet assay estimated higher tail intensities for 211At in contrast to 188Re. Compared with the complete repair (10%) after irradiation with 188Re, the radiotoxicity of alpha particles indicated reduced rejoining of DNA strand breaks (60-80% residual damage). Rejoining of DNA damage measured by the neutral comet method detected about 70% unrepaired strand breaks for 211At and 188Re. CONCLUSIONS There are major differences between the repair of strand breaks caused by 188Re and 211At detected by the alkaline comet assay. The DNA-damage induced by the high LET Emitter 211At remains nearly unrepaired detected by both alkaline and neutral comet assay. Represented data following irradiation of lymphocytes with alpha and beta particles demonstrated higher biological effectiveness of 211At by factors of 2.0-2.5.

Sodium-iodide symporter positive cells after intracellular uptake of 99mTc versus α-emitter 211At: Reduction of clonogenic survival and characterization of DNA damage

PURPOSE We evaluated the DNA damaging potential of Auger electrons emitted in the decay of (99m)Tc compared to α-particles of 211At. MATERIAL AND METHODS The impact of (99m)Tc and 211At was monitored in a NIS-expressing rat thyroid cell model PCCl3 with varying, yet defined intra- and extracellular radionuclide distribution (using ± perchlorate). The radiotoxicity of (99m)Tc and 211At was studied by the comet assay under neutral and alkaline conditions and colony formation. RESULTS In the presence of perchlorate, the radioactivity yielding 37% cellular survival, A37, was estimated to be (0.27 ± 0.02) MBq/ml and (450 ± 30) MBq/ml for 211At and (99m)Tc, respectively. In absence of perchlorate, cellular radiotracer uptake was similar for both radionuclides (2.2%, 2.7%), yet the A37 was reduced by 82% for the α-emitter and by 95% for (99m)Tc. Cellular dose increased by a factor of 5 (211At) and 38 (99mTc). Comet assays revealed an increased DNA damage after intracellular uptake of both radiotracers. CONCLUSIONS The data indicate damage to the cell to occur from absorbed dose without recognizable contribution from intracellular heterogeneity of radionuclide distribution. Comet assay under alkaline and neutral conditions did not reveal any shift to more complex DNA damage after radionuclide uptake. Cellular uptake of (99m)Tc and 211At increased cellular dose and reduced clonogenic survival.

Cellular damage in vitro - Influence of β-energy and intracellular radionuclide uptake: Influence of β-energy and intracellular radionuclide uptake

AIM The cellular damage of ionising radiation depends on dose, physical radiation quality (e. g. LET) and intracellular radionuclide uptake. The influence of two beta emitters (188Re and 131I) on the thyroid cell line PCCl3 was studied. Furthermore, we analysed the effect of intracellular accumulation. METHODS The thyroid cell line PCCl3 was irradiated with 188Re-perrhenate or 131I-sodium iodide in presence or absence of perchlorate. The initial DNA-damage was measured in the comet assay as olive tail moment (OTM). The colony forming assay detects the clonogenic cell survival as surviving fraction. Additional the intracellular radionuclide uptake was quantified. RESULTS Dose response curves were established for irradiation with 188Re-perrhenate or 131I-iodine under various extra- and intracellular activity distribution conditions. In the presence of perchlorate DNA-damage and clonogenic cell survival for both radionuclides were comparable. In the absence of perchlorate radionuclide uptake of 1.39% (131I) and 4.14% (188Re) were measured causing twofold higher radiotoxicity. Although 131I uptake was lower than 188Re uptake the OTM values were higher und surviving fractions were lower. CONCLUSIONS 131I, compared to 188Re, has lower mean beta energy and a higher LET, and therefore, it induced a higher DNA-damage even at lower intracellular uptake. An additional explanation for the higher radiotoxicity of 131I could be the higher dose exposition caused by cross-fire through neighborhood cells.