The aim of this article is to present a method for studying the shape of the dose and repair responses for X-ray-induced double-strand breaks (DSBs) as measured by neutral filter elution (NFE). The approach is closely related to a method we developed for the use of specific molecular size markers and used for determination of the absolute number of randomly distributed radiation-induced DSBs by pulsed-field gel electrophoresis (PFGE). Mouse leukemia L1210 cells were X-irradiated with 0-50 Gy. Samples were then evaluated both with PFGE and with NFE. Assuming that with both migration (PFGE) and elution (NFE), a heterogeneous population of double-stranded DNA fragments will start with the smallest fragments and proceed with increasingly larger fragments, it is possible to match the migration behavior of fractions of fragments smaller than a certain size to the fraction eluted at a specific time. This assumption does not exclude the possibility of DNA being sheared in the NFE filter. The yield, as determined by the size markers in PFGE, was used to find the corresponding elution times in the NFE experiment. These experimentally used elution times could then reversely be interpreted as size markers which finally were used to calculate DSBs/Mbp as a function of X-ray dose. The resulting lines were almost straight. The data were also plotted as relative elution and showed that, as expected, the dose response then appears with a more pronounced sigmoid shape. (C) 2003 by Radiation Research Society.

To estimate the effects of interaction of gamma-rays and an epoxide, cell survival and induction of DNA double-strand breaks (DSBs) following combined exposure to ionizing radiation and ethylene oxide (EtO) or propylene oxide (PO) were studied in human fibroblasts. Two treatment protocols were applied: (a) the cells were pre-exposed to different doses of gamma-rays and then treated with epoxide, and (b) the cells were pretreated with epoxide and then exposed to different doses of gamma-rays. Here we show that order of the treatment did not play a role in cell survival and that the effect of combined exposure on cell killing was additive for both epoxides. As to DNA DSBs induction, however, a difference dependent upon the order of the treatment was observed. While EtO or PO treatment followed by gamma-rays exposure led to an increased number of DSBs at higher gamma-ray doses (2-3 Gy), no significant increase of DSBs was detected after the opposite order of the treatment (gamma-ray exposure followed by EtO or PO treatment).

ESR spectroscopy is currently taking root as a key technology in dosimetry, dating and imaging. In dosimetry, it competes with cytometry in the fields of biological dosimetry and retrospective dosimetry, leads in high-level reference and routine dosimetry, is high-ranking among the methods to identify radiation preserved foods, represents a method of choice to date geological, archaeological and paleontological materials back millions of years, and has demonstrated capacity for imaging. Further scientific and technological progress as predicted in the recent past (Appl. Radiat. Isot. 52 (2000) 1023) is reviewed here. Additionally, the review is expanded to include international reports and recommendations on ESR dosimetry and dose reconstruction, under way at the American Society for Testing and Materials (ASTM), the International Organisation of Standards (ISO), the International Atomic Energy Agency (IAEA) and the International Commission on Radiation Units and Measurements (ICRU). Emphasis is placed on interpretation of tooth enamel doses in terms of organ and effective doses, using CT-based virtual humans. The future of EPR spectroscopy for in situ dose measurements is noted, depicting a non-destructive in vivo dosimetry applicable directly to individuals, but also to hominid and animal fossils for direct dating.

Purpose: Variation in sensitivity to radiotherapy among tumors has been related to the capacity of cells to repair radiation-induced DNA double-strand breaks (DSBs), DNA-dependent protein kinase (DNA-PK) and DNA ligases may affect DNA dsb rejoining. This study was performed to compare rate of rejoining of radiation-induced DSBs, DNA-PK, and DNA ligase activities in two human squamous carcinoma cell lines with different sensitivity to ionizing radiation.Methods and Materials: Cell survival of two human squamous carcinoma cell lines, UM-SCC-1 and UM-SCC-14A, was determined by an in vitro clonogenic assay. DSB rejoining was studied using pulsed field gel electrophoresis (PFGE), DNA-PK activity was determined using BIOTRAK DNA-PK enzyme assay system (Amersham), DNA ligase activity in crude cell extracts was measured using [5'-P-33] Poly (dA).(Oligo (dT) as a substrate. Proteolytic degradation of proteins was analyzed by means of Western blotting.Results: Applying the commonly used linear-quadratic equation to describe cell survival, S = e(-alpha D-beta D2), the two cell lines roughly have the same alpha value (similar to 0.40 Gy(-1)) whereas the beta value was considerably higher in UM-SCC-14A (0.067 Gy(-2) +/- 0.007 Gy(-2) [SEM]) as compared to UM-SCC-1 (0.013 Gy(-2) +/- 0.004 Gy(-2) [SEM]), Furthermore, UM-SCC-1 was more proficient in rejoining of X-ray-induced DSBs as compared to UM-SCC-14A as quantified by PFGE, The constitutive level of DNA-PK activity was 1.6 times higher in UM-SCC-1 as compared to UM-SCC-14A (p < 0.05), The constitutive level of DNA ligase activity was similar in the two cell lines.Conclusions: The results suggest that the proficiency in rejoining of DSBs is associated with DNA-PK activity but not with total DNA ligase activity, (C) 1998 Elsevier Science Inc.

The repair kinetics of DNA single- and double-strand breaks (SSBs, DSBs) induced with two carcinogenic epoxides, propylene oxide (PO) and epichlorohydrin (ECH), was studied in human diploid fibroblasts. The methods used were: alkaline DNA unwinding (ADU), the comet assay, and pulsed field gel electrophoresis (PFGE). About 70% of SSBs, measured by ADU, were rejoined after the treatment with 5 mMh and 10 mMh of PO within 20 hr, and the half-life was estimated to be ∼15 hr. On the other hand, effective rejoining of SSBs after ECH treatment was observed only at a dose of 1 mMh (a half-life of ∼15 hr), whereas after 2 mMh treatment, only 26% of SSBs could be rejoined within 20 hr. Furthermore, the use of the comet assay demonstrated that DNA strand breaks were effectively rejoined after PO and ECH treatment at doses of 5–10 mMh and 0.5–1 mMh, respectively. About 76% and 83% of DSBs induced by 5 and 10 mMh of PO, respectively, were rejoined within 4 hr after the treatment (a half-life of ∼2.5 hr), with little further repair thereafter. At lower dose of ECH (1 mMh) a half-life for DSBs rejoining was estimated to be ∼2 hr; however, only 29% of DSBs were rejoined within 2 hr at the higher dose of 2 mMh. After 18 hr, the rejoining following treatment with a lower dose was negligible. At a higher dose, a rapid accumulation of DSBs was observed, probably as the result of cell death and DNA degradation. The results demonstrate the capability of human diploid fibroblasts to repair DNA SSBs and DSBs at low-to-moderate doses of the epoxides. A weak capacity to rejoin DNA strand breaks induced by higher doses of ECH may be a consequence of its higher DNA alkylation activity and approximately 10 times higher toxicity compared to PO. Environ. Mol. Mutagen. 32:223–228, 1998 © 1998 Wiley-Liss, Inc.

The induction of DNA strand breaks in human diploid fibroblasts (VH-10) was demonstrated after in vitro exposure with two carcinogenic epoxides, propylene oxide (PO) and epichlorohydrin (ECH). Alkaline DNA unwinding (ADU), pulsed field gel electropharosis (PFGE), and the comet assay were used to measure DNA single. (SSBs) and double-strand breaks (DSBs). A dose-dependent increase of DNA strand breaks, measured by ADU, was observed in the dose range 2.5-20 mMh of PO and 0.25-2 mMh of ECH. The dose-response of ECH was about five times higher compared with that of PO (211 vs. 41 SSBs. 100 Mbp-1.mMh-1). The induction rates of DSBs, measured by PFGE, were found to be 18 times higher for ECH compared to PO (4.8 and 0.27 DSBs.100 Mbp-1.mMh-1 for ECH and PO, respectively). Using these two methods, the SSBs/ DSBs ratio was estimated to be 148 for PO and 44 for ECH. The data obtained by the comet assay also demonstrated a dose-dependent ability of PO and ECH to induce DNA damage. It was found that ECH was about six times more effective as an inducer of DNA strand breaks compared to PO (200 and 32x100 Mbp-1.mMh-1 for ECH and PO, respectively). The SSBs/DSBs ratios calculated using comet assay and PFGE data were 125 for ECH and 41 for PO. In addition, ECH is about 10 times more toxic than PO with respect to survival. These properties of ECH can at least in part be explained by its higher chemical reactivity connected with a higher rate of DNA alkylation.

ELECTROPHORESISVolume 17, Issue 6 p. 1087-1093 Review Mathematical analysis of DNA fragment distribution models used with pulsed-field gel electrophoresis for DNA double-strand break calculations Dr. Tomas Radivoyevitch, Corresponding Author Dr. Tomas Radivoyevitch [email protected] Department of Biometry, Medical University of South Carolina, Charleston, SC, USADepartment of Biometry, Medical University of South Carolina, Charleston, SC 29425 USA (Tel: +803-766-7064; Fax: +803-792-0539;)===Search for more papers by this authorBjörn Cedervall, Björn Cedervall Departments of Medical Radiobiology and Medical Radiation Physics, Karolinska Institutet Stockholm, SwedenSearch for more papers by this author Dr. Tomas Radivoyevitch, Corresponding Author Dr. Tomas Radivoyevitch [email protected] Department of Biometry, Medical University of South Carolina, Charleston, SC, USADepartment of Biometry, Medical University of South Carolina, Charleston, SC 29425 USA (Tel: +803-766-7064; Fax: +803-792-0539;)===Search for more papers by this authorBjörn Cedervall, Björn Cedervall Departments of Medical Radiobiology and Medical Radiation Physics, Karolinska Institutet Stockholm, SwedenSearch for more papers by this author First published: 1996 https://doi.org/10.1002/elps.1150170618Citations: 12AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 Tobias, C. A., Radiat. Res. 1985, 104, S77– S95. 2 Ostashevsky, J. Y., Radiat. Res. 1989, 118, 437– 466. 3 Cedervall, B., Wong, R., Albright, N., Dynlacht, J., Lambin, P., Dewey, W. C., Radiat. Res. 1995, 143, 8– 16. 4 Cedervall, B., Källman, P., Dewey, W. C., Radiat. Res. 1995, 142, 23– 28. 5 Contopoulou, C. R., Cook, V. E., Mortimer, R. K., Yeast 1987, 3, 71– 76. 6 Cook, V. E., Mortimer, R. K., Radiat. Res. 1991, 125, 102– 106. 7 Lawrence, T. S., Normolle, D. P., Davis, M. A., Maybaum, J., Int. J. Radiat. Oncol., Biol., Phys. 1993, 27, 659– 663. 8 Cedervall, B., Wong, R., Albright, N., Dynlacht, J., Lambin, P., Dewey, W. C., Radiat. Res. 1995, 144, 122. 9 Lehmann, A. R., Ormerod, M. G., Biochim. Biophys. Acta 1970, 204, 128– 143. 10 Cedervall, B., Radivoyevitch, T., Electrophoresis 1996, 17, 1078– 1084. 11 Friedl, A. A., Beisker, W., Hahn, K., Eckhardt-Schupp, F., Kellerer, A. M., Int. J. Radiat. Biol. 1993, 63, 173– 181. 12 Kraxenberger, A., Friedl, A. A., Kellerer, A. M., Electrophoresis 1994, 15, 128– 136. Citing Literature Volume17, Issue61996Pages 1087-1093 ReferencesRelatedInformation

ELECTROPHORESISVolume 17, Issue 6 p. 1080-1086 Review Methods for analysis of DNA fragment distributions on pulsed field gel electrophoretic gels Dr. Björn Cedervall, Corresponding Author Dr. Björn Cedervall Departments of Medical Radiobiology and Medical Radiation Physics, Karolinska Institutet, Stockholm, SwedenDepartment of Medical Radiation Physics, Karolinska Institute, P.O. Box 260, S-171 76 Stockholm, Sweden (Tel: +468-729-4359; Fax: +46-8-34-35-25)===Search for more papers by this authorTomas Radivoyevitch, Tomas Radivoyevitch Department of Biometry, Medical University of South Carolina, Charleston, SC, USASearch for more papers by this author Dr. Björn Cedervall, Corresponding Author Dr. Björn Cedervall Departments of Medical Radiobiology and Medical Radiation Physics, Karolinska Institutet, Stockholm, SwedenDepartment of Medical Radiation Physics, Karolinska Institute, P.O. Box 260, S-171 76 Stockholm, Sweden (Tel: +468-729-4359; Fax: +46-8-34-35-25)===Search for more papers by this authorTomas Radivoyevitch, Tomas Radivoyevitch Department of Biometry, Medical University of South Carolina, Charleston, SC, USASearch for more papers by this author First published: 1996 https://doi.org/10.1002/elps.1150170617Citations: 14AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume17, Issue61996Pages 1080-1086 RelatedInformation

For theoretical reasons, it is incorrect to define experimentally the half-life times of DNA double-strand breaks (DSBs) as the half-life time of an amount of DNA. This is illustrated by one example of human DNA, where the half-life for first-order kinetics of the disappearance of DSBs has been assumed to be 10 min. Experimental sources of errors and their influence on experimental results are analyzed. Some experimental situations may lead to serious misinterpretation data. The differential decreases in fractions released (amounts of DNA) as often followed in pulsed-field gel electrophoresis depend on run conditions, background and level of DSB induction and are a function of time itself--a time function that is unrelated to the half-life of DSBs. It is shown that, using the decrease of a measured amount of DNA, one may obtain practically any value for the half-life time.

Pulsed-field gel electrophoresis has been applied to separate DNA from mouse L1210 cells exposed to X-ray doses of 1 to 50 Gy. Simultaneous separation of marker chromosomes in the range 0.1 to 12.6 Mbp allowed calculation of the size distribution of the radiation-induced fragments. The distribution was consistent with a random induction of double-strand breaks (DSBs). A theoretical relationship between the size distribution of such fragments and the average number of induced breaks was used to calculate the yield and dose response. The DNA distribution was determined by both radiolabeling and fluorescence staining. Two independent methods were used to evaluate the radiation-induced yield of DSBs, both assuming that all DNA is broken at random. In the first method we compared the theoretical and experimental fraction of DNA that is below a given size limit. By this method we estimated the yield to be 0.006-0.007 DSB/Gy per million base pairs using the radiolabel and 0.004-0.008 DSB/Gy per million base pairs by fluorescence staining. The dose response was linear in both cases. In the second method we looked only at the size distribution in the resolving part of the gel and compared it to the theoretical distribution. By this method a value of approximately 0.012 DSB/Gy/Mbp was found, using fluorescence as a measure of DNA distribution. In a normal diploid mammalian genome of size 6000 Mbp, this is equivalent to a yield of 25-50 DSBs/Gy or 70 DSBs/Gy, respectively. The second approach, which looks only at the smaller fragments, may overestimate the yield, while the first approach suffers from uncertainties about the fraction of DNA irreversibly trapped in the well. The assay has the capacity to detect a dose of less than 1 Gy.

The aim of this work was to measure simultaneously and in a quantitative manner double-strand breaks (DSBs), interphase chromosome breaks and cell lethality either immediately after irradiation, or at various times thereafter (up to 24 h), in cells of three nontransformed human fibroblast cell lines of widely different intrinsic radiosensitivity. We wished to assess initial damage, repair kinetics and residual damage at the DNA and the chromosome level, and to correlate these parameters with cell killing. We employed HF19 cells, a normal fibroblast cell line, AT2 cells, a radiosensitive cell line from a patient suffering from ataxia telangiectasia (AT), and 180BR cells, a radiosensitive cell line from a patient with no clinical symptoms of AT. AT2 and 180BR cells, in addition to being radiosensitive, also display a reduced ability to repair potentially lethal damage compared to HF19 cells. The yield of DSBs, as measured by pulsed-field gel electrophoresis, is similar in all three cell lines (slopes correspond to 1.6-1.7% Gy-1 of DNA-associated radioactivity released from the gel well into the lane). In contrast, residual DSBs measured 24 h after irradiation are almost zero for HF19 cells (0.1% confidence interval = 0-1.4%), but are 12.5% (+/- 2.3%) and 43.8% (+/- 1.2%) of those measured immediately after irradiation in AT2 and 180BR cells, respectively. Residual interphase chromosome breaks are 11.6% (+/- 1.6%), 29.7% (+/- 5.7%) and 41.4% (+/- 2.2%) of those measured immediately after irradiation in HF19, AT2 and 180BR cells, respectively. Neither the initial yield of DSBs nor that of excess interphase chromosome breaks can explain the differences in radiosensitivity between the three cell lines; however, there is a correlation between residual DSBs, rate of DSB rejoining at 24 h, residual interphase chromosome breaks on the one hand and cell survival on the other hand.

Different methods were used for evaluating data for DNA double-strand breaks (DSBs), as obtained by pulsed-field gel electrophoresis (PFGE) after X irradiation of Chinese hamster ovary cells. A total of 60 data points in the dose range of 0 to 116 Gy, along with repair data for 30 and 60 Gy, were analyzed by four methods: (1) percentage of DNA released from the plug, (2) specific size markers (percentage of DNA less than specific sizes, (3) fragment size distributions and (4) shape of the molecular weight (M) distributions. With the last method, both the slope and the intercept of the logarithm of the amount of radioactive DNA/delta M/M plotted as a function of M were used for calculating DSBs/100 Mbp. The slope and the intercept analyses differ in that the former is relatively independent of DNA trapped in the agarose plugs, i.e. cannot be released by doses of 100-150 Gy, whereas the intercept is dependent on the percentage of DNA trapped. Also, calculations of DSBs/100 Mbp for methods 1, 2 and 3 depend on the amount of DNA trapped in the plug. However, the slope method is unreliable for doses below about 20 Gy, and the scatter of data points is much greater than that obtained by the intercept method and by methods 1, 2 and 3. Therefore, the fragment size distribution and the specific size marker methods give the most consistent results, with 0.49 +/- 0.03 (95% CI) (DSBs/100 Mbp)/Gy. With the specific size marker method, however, care must be taken in selection of size markers in relation to the levels of DSBs of interest. Assuming randomly distributed DSBs, all four methods gave essentially the same results; i.e., the dose response was linear with a calculated level of 0.5-0.6 (DSBs/100 Mbp)/Gy, which is the same as 0.47-0.62 determined previously by calibrating with 125IdU.

For a given amount of cell killing, heat alone (10-80 min, 45.5 degrees C) induced very few double-strand breaks (dsbs) compared with X-rays. Furthermore, 10 min at 45.5 degrees C immediately prior to X-rays caused only a 1.3-fold increase in the slope of the X-ray-induced dsb dose-response curve, i.e. 0.67 +/- 0.006 (95% confidence) dsbs/100Mbp/Gy for heated cells compared with 0.53 +/- 0.005 for unheated control cells. However, this same heat treatment caused > 5-fold inhibition in the rate of repair of dsbs induced by 60-Gy X-rays, with the degree of inhibition being much less in thermotolerant (TT) cells than in non-tolerant (NT) cells. This reduced inhibition of repair in TT cells correlated with the more rapid removal of excess nuclear protein from nuclei isolated from TT cells than from NT cells. These results plus a TT ratio of 2-3 for both heat-induced radiosensitization and heat-inhibition of repairing dsbs are consistent with the hypothesis that heat radiosensitization results primarily from heat aggregation of nuclear protein interfering with access of repair enzymes to DNA dsbs. The selective heat-radiosensitization of S-phase cells, however, may result from an increase in radiation-induced dsbs in or near replicating regions. For example, a preferential increase in dsbs in replicating DNA compared with bulk DNA was found following either hyperthermia alone (10-30 min, 45.5 degrees C) or a combined treatment (10 min, 45.5 degrees C before 60 Gy). A 30-min treatment at 45.5 degrees C induced dsbs equivalent to approximately 10 Gy in replicating DNA compared with 3-5 Gy in bulk DNA. When cells were heated immediately before irradiation, the increase in dsbs induced in the replicating DNA by 60 Gy was equivalent to 200 Gy. We hypothesize that the observed 2-fold increase in single-stranded regions in replicating DNA after heat resulted in radiation selectively inducing dsbs at or near the replication fork where the heat-induced increase in single-stranded DNA should occur. Thus, this preferential increase in dsbs in the replicating DNA by heat alone and especially when heat was combined with radiation may explain at least in part, the high sensitivity of S-phase cells to heat killing and heat radiosensitization.

Internucleosomal DNA fragmentation (DNA laddering) and formation of apoptotic bodies have long been considered characteristic features of apoptosis. However, recent work has shown that formation of high molecular weight DNA fragments precedes internucleosomal cleavage and may involve mechanisms that differ from those responsible for DNA laddering. Here, we show that glucocorticoid treatment of human thymocytes stimulated the formation of high molecular weight DNA fragments by Ca(2+)- and endonuclease-mediated mechanisms. Either the removal of Ca2+ from the medium or pretreatment of the cells with the intracellular Ca2+ chelator, BAPTA-AM, prevented the formation of large DNA fragments. Further, treatment of the thymocytes with the microsomal Ca(2+)-ATPase inhibitor, thapsigargin, which caused a sustained increase in intracellular Ca2+ concentration, was in itself sufficient to activate high molecular weight DNA fragmentation. Our results show that Ca(2+)-dependent mechanisms promote the multistep chromatin cleavage in human thymocyte apoptosis.

Cells of a small cell lung carcinoma cell line, U-1285, and an undifferentiated large cell lung carcinoma cell line, U-1810, differ in radiosensitivity in parallel to the clinical radiosensitivity of the kind of tumors from which they are derived. The surviving fraction at 2 Gy (SF2) was 0.25 that of U-1285 cells and 0.88 that of U-1810 cells. We investigated the induction of DNA double-strand breaks (DSBs) by X rays and DSB rejoining in these cell lines. To estimate the number of DSBs we used a model adapted for pulsed-field gel electrophoresis (PFGE). The induction levels were of the same magnitude (0.46 and 0.51 DSB/100 Mbp/Gy for U-1810 and U-1285 cells, respectively). These levels of induction do not correlate with radiosensitivity as measured by cell survival assays. Rejoining of DSBs after doses in the range of 0-50 Gy was followed for 0, 15, 30, 60 and 120 min. We found a difference in the velocity of repair during the first hour after irradiation which is parallel to the differences in radiosensitivity. Thus U-1810 cells exhibit a fast component of repair, with about half of the DSBs being rejoined during the first 15 min, whereas U-1285 cells lack such a fast component, with only about 5% of the DSBs being rejoined after the same time. In addition there was a numerical albeit not statistical difference at 120 min, with more residual DSBs in the U-1285 cells compared to the U-1810 cells.

In vitro exposure of normal human diploid fibroblasts (strain VH-10) to ethylene oxide (EtO) induced DNA strand breaks in the dose range of 2.5-30 mMh of EtO. Alkaline DNA unwinding (ADU), neutral filter elution (NFE), pulsed field gel electrophoresis (PFGE), and the comet assay were used to measure DNA single (SSBs) and double strand breaks (DSBs). Different induction rates of SSBs and DSBs, depending on applied method and also on treatment conditions (cells in monolayer or in suspension were used), were found. A dose-dependent increase of DNA strand breaks was found by the ADU method in the dose range of 2.5-20 mMh of EtO when treatment was performed in monolayer and in suspension. DSBs were detected by NFE only when the cells were treated with EtO in suspension (doses 10-30 mMh). The highest induction rate of DSBs (about 4 DSBs per 100 Mbp per 1 mMh of EtO) was detected in suspension with PFGE applied. We have shown that heat-labile sites are formed by EtO. Presumably, the different DSB levels detected by PFGE and NFE result from the conversion of these sites to DSBs during cell lysis at elevated temperature in the PFGE method. The results of the comet assay confirmed that apoptotic processes are not involved in the formation of DSBs in our experimental conditions (less than 1% of apoptotic cells were observed at all doses studied). Possible mechanisms for the induction of DNA strand breaks by EtO-treatment are discussed. The capacity to repair DSBs in EtO-exposed (5-7.5 mMh) cells was studied, and it was found that a considerable part of the damage (about 50%) could be repaired during 18 hr of incubation.