Measurement Of DNA Double-strand Breaks Research Articles

The cytotoxicity and genotoxicity of hexavalent chromium in medaka ( Oryzias latipes) cells

Chromium is an increasing health concern for aquatic environments, however, the mechanism of chromium toxicity in aquatic species is yet unknown. We used a medaka ( Oryzias latipes) fin cell line to investigate the cytotoxicity and genotoxicity of sodium chromate, a soluble form of hexavalent chromium. We used a clonogenic cytotoxicity assay to measure sodium chromate cytotoxicity, gamma-H2A.X immunofluoresence to measure DNA double-strand breaks, and chromosome damage to measure clastogenicity. We found that sodium chromate is cytotoxic to medaka fin cells, with toxicity increasing in a concentration-dependent manner. Treatments of 0.5, 1, 5, 10, 25, 50 and 100 μM sodium chromate caused 100, 103.5, 87.8, 77.5, 40.9, 15 and 2.7% survival, respectively, relative to the control. We visualized DNA double-strand breaks in medaka cells through the formation of gamma-H2A.X foci. Breaks could be detected at concentrations as low as 1 μM. We also found that sodium chromate induces chromosomal aberrations, causing chromatid lesions and exchanges that increase with concentration. Treatments of 0, 1, 5, 10 and 25 μM sodium chromate damaged 10.3, 17, 32.3, 43 and 51.6% of metaphases and induced 13, 23, 44, 69 and 118 total aberrations in 100 metaphases, respectively. These data show that hexavalent chromium is both cytotoxic and genotoxic to fish cells. Our results set the context for future work in the medaka cell culture model and provide important tools for investigating mechanisms of toxicity in aquatic organisms.

Carcinogen-Induced DNA Double Strand Break Repair in Sporadic Breast Cancer

Induction of DNA double strand breaks and alterations in the repair of these breaks is implicated in breast carcinogenesis. Prior studies have demonstrated that peripheral blood mononuclear cells (PBMC) from breast cancer patients exhibit increased numbers of DNA strand breaks after exposure to ionizing radiation, but these studies did not specifically measure DNA double strand breaks and it is not known whether chemical carcinogens produce similar effects. PBMC from 32 women undergoing breast surgery were genotyped at nine loci of seven DNA repair genes. DNA double strand break repair was measured using the neutral comet assay after exposure to ionizing radiation (0.5 Gy) or bioactivated benzo[a]pyrene (B[a]P, 5 microM. PBMC from breast cancer patients showed higher levels of residual DNA double strand breaks 30 min after exposure to radiation than PBMC from patients with benign breast disease (1.40 times baseline [95% confidence intervals [CI] 1.29-1.51] versus 1.24 times baseline [95% CI 1.15-1.33], respectively, P = 0.04). The response to B[a]P trended in the same direction, but did not reach statistical significance. The MGMT K178R variant genotype was associated with improved DNA double strand break repair in PBMC exposed to B[a]P. Reduced repair of radiation-induced DNA double strand breaks in PBMC is a robust biomarker of breast cancer risk. Reduced DNA repair capacity may have a genetic component even in sporadic breast cancer.

A non-radioactive, PFGE-based assay for low levels of DNA double-strand breaks in mammalian cells

A PFGE method was adapted to measure DNA double-strand breaks (DSBs) in mammalian cells after low (0–25 Gy) doses of ionising radiation. Instead of radionuclide incorporation, DNA staining in the gel by SYBR-Gold was used, which lowered the background of DNA damage and could be applied to non-cycling cells. DSB level was defined as a product of a fraction of DNA released to the gel (FR) and a number of DNA fragments in the gel (DNA fragm) and expressed as a percentage above control value. The slope of the dose-response curve was two-fold higher compared to that with FR alone as DSB level indicator (31.4 versus 15.6% per Gy). Two alternative ways were proposed to determine the total amount of DNA, used for FR calculation: measurement of DNA content in a plug not subjected to electrophoresis, with the use of Pico-Green, or estimation of DNA released to the gel from a plug irradiated with 600 Gy of γ-rays. The limit of DSB detection was 0.25 Gy for human G1-lymphocytes and 0.5–1 Gy for asynchronous cultures of human glioma M059 K and J or mouse lymphoma L5178Y-R and -S cells. Specificity of our PFGE assay to DSB was confirmed by the fact that no damage was detected after treatment of the cells with H 2O 2, an inducer of single-strand DNA breaks (SSBs). On the contrary, the H 2O 2 inflicted damage was detected by neutral comet assay, attaining 160% above control (equivalent to 2.5 Gy of X-radiation). DSB rejoining, measured in cells after X-irradiation with a dose of 10 Gy, generally proceeded faster than that measured previously after higher (30–50 Gy) doses of ionising radiation. Clearly seen were defects in DSB rejoining in radiosensitive M059 J and L5178Y-S cells compared to their radioresistant counterparts, M059 K and L5178Y-R. In some cell lines, a secondary post-irradiation increase in DSB levels was observed. The possibility is considered that these additional DSBs may accumulate during processing of non-DSB clustered DNA damage or/and represent early apoptotic events.

Comments on 'Underestimation of the small residual damage when measuring DNA double-strand breaks (DSB): is the repair of radiation-induced DSB complete?'

Comments on 'Underestimation of the small residual damage when measuring DNA double-strand breaks (DSB): is the repair of radiation-induced DSB complete?'

Underestimation of the small residual damage when measuring DNA double-strand breaks (DSB): is the repair of radiation-induced DSB complete?

Purpose: To overcome the underestimation of the small residual damage when measuring DNA double-strand breaks (DSB) as fraction of activity released (FAR) by pulsed-field gel electrophoresis. Materials and methods: The techniques used to assess DNA damage (e.g. pulsed-field gel electrophoresis, neutral elution, comet assay) do not directly measure the number of DSB. The Blocher model can be used to express data as DSB after irradiation at 4° C by calculating the distribution of all radiation-induced DNA fragments as a function of their size. We have used this model to measure the residual DSB (irradiation at 4° C followed by incubation at 37° C) in untransformed human fibroblasts. Results: The DSB induction rate after irradiation at 4° C was 39.1 +/- 2.0Gy -1. The DSB repair rate obtained after doses of 10 to 80Gy followed by repair times of 0 to 24h was expressed as unrepaired DSB calculated from the Blocher formula. All the damage appeared to be repaired at 24h when the data were expressed as FAR, whereas 15% of DSB remained unrepaired. The DSB repair rate and the chromosome break repair rate assessed by premature condensation chromosome (PCC) techniques were similar. Conclusion: The expression of repair data in terms of FAR dramatically underestimates the amount of unrepaired DNA damage. The Blocher model that takes into account the size distribution of radiation-induced DNA fragments should therefore be used to avoid this bias. Applied to a normal human fibroblast cell line, this model shows that DSB repair is never complete.

Comparison between Pulsed-Field Gel Electrophoresis and the Comet Assay as Predictive Assays for Radiosensitivity in Fibroblasts

The radiosensitivity of skin fibroblasts derived from patients as measured in vitro by a clonogenic survival assay appears to correlate with the risk of developing severe late reactions to radiation. Unfortunately, these assays are clinically impractical as a predictive test for radiosensitivity. The purpose of this study was to assess the utility of two possible surrogate assays for radiosensitivity, pulsed-field gel electrophoresis (PFGE) and single-cell gel electrophoresis (comet assay), both of which can be used to measure DNA double-strand breaks. Twenty-three nontransformed human fibroblast cell lines exhibiting a range of radiosensitivities were studied with both of these assays. The results were correlated with measurements of radiosensitivity obtained as part of a larger study examining the correlation between cellular radiosensitivity and clinical response. [2-(14)C]Thymidine-labeled confluent cultures were irradiated at 1.0 Gy/min with doses of 0 to 150 Gy. After allowing 4 h for repair at 37 degrees C, cells were trypsinized and aliquots were used for preparing slides for the comet assay. After neutral lysis and electrophoresis, the slides were stained with ethidium bromide and 50 comet moments were measured for each dose. The remainder of the cells were formed into agarose plugs and, after neutral lysis, were subjected to PFGE. The fraction of activity released (FAR) from the well was measured by scintillation counting of appropriate segments of each gel lane. Cellular radiosensitivity was measured with a standard clonogenic assay at a low dose rate of 1.2 cGy/min, and the dose that resulted in a surviving fraction of 0.01 (D0.01) was calculated. The slope of the plot of comet moment as a function of dose for each cell line did not correlate with D0.01 (R = 0.36, P > 0.1). In contrast, the slope of the FAR as a function of dose had a weak inverse correlation with D0.01 (R = 0.43 and P = 0.05) such that the more radiosensitive cell lines exhibited a steeper dose response for FAR. Although the correlation between the slope of the dose response for FAR and D0.01 was weak, refinement of the PFGE technique may provide a potentially useful predictive assay for radiosensitivity.

SYBR Green I staining of pulsed field agarose gels is a sensitive and inexpensive way of quantitating DNA double-strand breaks in mammalian cells.

Pulsed field gel electrophoresis (PFGE) is widely used to measure DNA double strand breaks (dsb). The DNA of cultured cells can be prelabelled with radioactivity, which helps greatly in detection and quantitation of DNA dsb. However, this approach cannot be used with non-cycling cells from biopsy material. We describe a method which uses SYBR Green I to stain DNA in dried agarose gels. DNA is detected and analysed using readily available camera equipment and image analysis software. This method is as sensitive as [3H]thymidine prelabelling of cells and allows DNA dsb to be measured simply and economically in non-cycling cells.

Use of pulsed-field gel electrophoresis to investigate factors influencing the measurement of DNA double-strand breaks in human brain tumour specimens.

We used pulsed-field gel electrophoresis combined with densitometry to investigate variables influencing the measurement of DNA double-strand breaks (dsb) in human brain tumour cells and frozen tissue. Our studies showed that this system worked best when we ran the gel at 60 V for 22-25 h at 18 degrees C with a pulse time of 15 s in 1xTris-acetic acid-EDTA buffer. Because the densitometric analysis worked well only when DNA concentrations were within a certain range, we developed a quick assay using a DNA-specific dye (TO-PRO-1) to determine concentrations before making agarose plugs. When we used our optimal procedure to measure dsb in six frozen human brain tumour specimens, we found that radiation-resistant tumours had significantly more initial dsb than did radiation-sensitive tumours. The number of residual dsb could not be ascertained because the process of freezing the specimens destroyed dsb repair ability.

Measurement of DNA Double-Strand Breaks in Mammalian Cells by Pulsed-Field Gel Electrophoresis: A New Approach Using Rarely Cutting Restriction Enzymes

A new method is presented for measuring DNA double-strand breaks (DSBs) which combines the technique of pulsed-field gel electrophoresis (PFGE) with the idea of velocity sedimentation. Purified DNA samples were treated with restriction enzymes, which results in DNA of sizes which can be separated by PFGE. After electrophoresis, unirradiated DNA shows a size distribution (obtained with the help of a specially developed software program) similar to that obtained with the sedimentation technique; with X irradiation, this distribution is shifted to lower molecular weight with increasing dose. The rate of DSB induction was calculated by comparing the curves obtained experimentally with theoretical distributions (based on the assumption that breaks are formed according to Poisson statistics). The method was tested by measuring X-ray-induced DSBs in P3 (derived from human epithelial teratoma) cells. The induction of DSBs was found to be linear with dose and a rate of 5.4 x 10(-3)/Mbp/Gy was obtained.

On the linearity of the dose-effect relationship of DNA double strand breaks.

Most radiation biologists believe that DNA double-strand breaks are induced linearly with radiation dose for all types of radiation. Since 1985, with the advent of elution and gel electrophoresis techniques which permit the measurement of DNA double-strand breaks induced in mammalian cells at doses having radiobiological relevance, the true nature of the dose-effect relationship has been brought into some doubt. Many investigators measured curvilinear dose-effect relationships and a few found good correlations between the induction of the DNA double-strand breaks and cell survival. We approach the problem pragmatically by assuming that the induction of DNA double-strand breaks by 125I Auger electron emitters incorporated into the DNA of the cells is a linear function of the number of 125I decays, and by comparing the dose-effect relationship for sparsely ionizing radiation against this standard. The conclusion drawn in that the curvilinear dose-effect relationships and the correlations with survival are real. The problem of why the dose-effect relationship is curvilinear when microdosimetric considerations predict that even for sparsely ionizing radiation energy deposition in and close to the Watson-Crick double helix should give a linear dose-effect relationship remains.

Comparison between Pulsed-field and Constant-field Gel Electrophoresis for Measurement of DNA Double-strand Breaks in Irradiated Chinese Hamster Ovary Cells

Pulsed-field gel electrophoresis (PFGE) is one of the most sensitive methods for detecting DNA double-strand breaks in mammalian cells. However, it has been observed that constant-field gel electrophoresis (CFGE), when optimized, can detect breaks with equal efficiency. The migration of DNA from the well and the separation of DNA molecules according to size appear to be different processes; only the latter requires the application of PFGE. CFGE is very sensitive and can detect DNA damage produced by less than 5 Gy of radiation. Low voltage (ca. 0.6 V/cm) during electrophoresis appears to be essential for the migration of the largest fraction of DNA from the agarose plug containing the cells; the electrophoresis run time, cell density in the plug, agarose concentration, nature of detergent and extent of radiolabelling are less important. It is concluded that CFGE is equally sensitive but more rapid and economical than PFGE for the measurement of DNA damage.

Measurement of DNA double-strand breaks in CHO cells at various stages of the cell cycle using pulsed field gel electrophoresis: calibration by means of 125I decay.

Experiments were performed to calibrate a recently developed pulsed field gel electrophoresis assay, the asymmetric field inversion gel electrophoresis (AFIGE), for the measurement of double-strand breaks (dsb) in the DNA of mammalian cells. Calibration was carried out by means of 125I decay accumulation under conditions preventing repair, and is based on the observation that each 125I decay in the DNA produces approximately one dsb. Iodine was incorporated into DNA in the form of 5'-iododeoxyuridine and decay accumulation was allowed in cells kept frozen at -80 degrees C. Since widely different DNA damage dose-response curves were obtained in cells exposed to X-rays in various phases of the cell cycle, calibration was performed using synchronized populations of cells that were allowed to accumulate DNA damage in G1, G1/S, mid-S, and G2 + M. For this purpose the fraction of activity (in DNA) released from the plug (FAR) was measured and correlated to the number of 125I decays accumulated during the elapsed period of time. Fluctuations in the FAR per 125I decay were observed throughout the cell cycle that were similar to those previously reported for the FAR per Gy of X-rays. Comparison of the FAR per 125I decay with the FAR per Gy gave an induction of 21 +/- 3, 31 +/- 3, 21 +/- 3 and 26 +/- 8 dsb per Gy per diploid DNA complement for G1, G1/S, S, and G2 + M cells, respectively. The results suggest that the observed fluctuations in the FAR per Gy throughout the cycle reflect cell-cycle-associated differences in the physicochemical properties of the DNA molecules that alter their electrophoretic mobility, rather than variations in the induction of dsb per Gy, i.e. the sensitivity of the assay fluctuates throughout the cycle. We propose that similar phenomena underlie the observed fluctuations throughout the cell cycle in the fraction of activity eluted per Gy in the non-unwinding filter elution assay. 125I decays accumulated at 4 degrees C in partly purified DNA from cells embedded in agarose plugs and lysed immediately, gave FAR identical to those obtained with cells kept frozen. This finding suggests that indirect effects do not significantly contribute to DNA damage induction by 125I decay, and indicate that calibration of electrophoresis techniques for dsb measurements can be carried out using this simplified protocol.

Measurement of Radiation-Induced DNA Double-Strand Breaks by Pulsed-Field Gel Electrophoresis

We have examined the use of pulsed-field gel electrophoresis (PFGE) to measure DNA double-strand breaks induced in CHO cells by ionizing radiation. The PFGE assay provides a simple method for the measurement of DNA double-strand breaks for doses as low as 3-4 Gy ionizing radiation, and appears applicable for the measurement of damage produced by any agent producing double-strand breaks. The conditions of transverse alternating field electrophoresis determined both the sensitivity of the assay and the ability to resolve DNA fragments with different sizes. For example, with 0.8% agarose and a 1-min pulse time at 250 V for 18 h of electrophoresis, 0.39% of the DNA per gray migrated into the gel, and only molecules less than 1500 kb could be resolved. With 0.56% agarose and a 60-min pulse time at 40 V for 6 days of electrophoresis, 0.55-0.90% of the DNA per gray migrated into the gel, and molecules between 1500 and 7000 kb could be resolved.

On the Measurement of DNA Double-Strand Breaks by Neutral Elution

Neutral elution is a sensitive and convenient method for measuring double-strand breaks in cellular DNA, but results obtained with this method are controversial, particularly as regards form of dose-response relationship. We pointed out in a recent publication (1) that explanation for different results obtained by neutral elution compared with neutral sedimentation remains obscure. The possibility exists that types of radiation damage other than double-strand breaks might affect neutral elution behavior. In a recent Letter to Editor, Hutchinson (2) stated that single-strand breaks might alter neutral elution because of configurational changes in DNA and/or increased susceptibility to shear but the necessary experiments to determine effect of single-strand breaks on neutral elution have not been performed. These necessary experiments must test whether single-strand breaks can modulate elution behavior of DNA containing a significant number of double-strand breaks, and we have performed such experiments. We induced double-strand breaks in DNA of human P3 epithelioid cells using 125I decay exactly as described previously (3), then, after allowing for accumulations of specific numbers of decays, we induced various numbers of single-strand breaks in cells by using treatments known to produce many single-strand breaks and few or no double-strand breaks: H202 (4) and bromodeoxyuridine photolysis (5), as well as y radiation. In no case (three measurements for each single-strand breakinducing treatment at different levels of induced singleand double-strand breaks) did extra single-strand breaks have any measurable effect on elution profiles (manuscript in preparation). Thus we have experimental evidence that neutral elution is not affected by concomitant singlestrand breaks, and this explanation for discrepancy between neutral sedimentation and neutral elution appears to be invalid. With respect to effect of protein crosslinked to DNA, more rigorous DNA lysis and cleaning procedures were described by Okayasu and Iliakis (6), who obtained double-strand break dose responses without a shoulder and, in some cases, straight elution profiles following these treatments. We have repeated those experiments as closely as possible using P3 cells, but saw neither exponential profiles nor dose responses without shoulders that Okasayu and Iliakis obtained. Therefore, DNA-to-protein crosslinking does not appear to be a determinant of neutral elution behavior in all types of cells. Thus question of different form of dose response measured by elution and sedimentation is still not resolved. Hutchinson's critique of neutral elution did not mention several positive aspects of assay or generally negative aspects of neutral sedimentation covered very thoroughly by van der Schans (7). The validity of elution assay is supported by fact that singleto doublestrand break ratios caused by different agents (which may differ by more than two orders of magnitude) detected by neutral elution correspond well with results obtained by other methods. Neutral sedimentation is beset with problems of irreproducibility caused in part by susceptibility of DNA to shear (7), as well as insensitivity. (Equivalent sensitivity to that of neutral elution has been achieved (8) but only by using extraordinarily laborious sample-handling procedures and 4-day sedimentation times.) Although neutral sedimentation, unlike neutral elution, is based upon mathematical theory, extent to which sedimentation theory is both untested and problematic in range of molecular weights relevant to low-dose studies in mammalian cells (9) should not be ignored. Despite unresolved probms, neutral elution remains only practical means of determining double-strand breaks at relatively low radiation doses.

Calibration of Pulsed Field Gel Electrophoresis for Measurement of DNA Double-strand Breaks

The pulsed field gel electrophoresis (PFGE) assay was calibrated for the measurement of X-ray-induced DNA double-strand breaks in Chinese hamster ovary (CHO) cells. Calibration was conducted by incorporating [125I]deoxyuridine into DNA, which induces one double-strand break for every disintegration that occurs in frozen cells. Based on the percentage of the DNA migrating into the gel, the number of breaks/dalton/Gy was estimated to be (9.3 +/- 1.0) x 10(-12). This value is close to (10 to 12) x 10(-12) determined by neutral filter elution using similar cell lysis procedures at 24 degrees C and at pH 8.0. Also, the estimate is in good agreement with the value of (11.7 +/- 2) x 10(-12) breaks/dalton/Gy as measured in Ehrlich ascites tumour cells using the neutral sucrose gradient method (Blöcher 1988), and (6 to 9) x 10(-12) breaks/dalton/Gy as measured in mouse L and Chinese hamster V79 cells using neutral filter elution (Radford and Hodgson 1985).

On the Measurement of DNA Double-Strand Breaks by Neutral Elution

DNA double-strand breaks unquestionably increase the rate at which DNA is eluted from cell lysates on a filter, in the neutral elution assay introduced by Bradley and Kohn in 1979 (1). Double-strand breaks are generally agreed to be one of the most biologically important types of DNA damage formed by ionizing radiation, and the neutral elution assay has been widely used to detect such breaks. The purpose here is to bring attention to the possibility that neutral elution may not always give a useful measure of the number of double-strand breaks. This letter may be regarded as a follow-up to a comment by Gunner Ahnstrom in 1985 (2), which has not received sufficient attention. If is it assumed that the rate of DNA elution under nondenaturing conditions is controlled fundamentally by double-strand breaks, then according to several studies, the number varies with dose D of ionizing radiation as aD + fD2 (3-9). The presence of the quadratic term is confirmed in neutral elution measurements on cells irradiated by ionizing radiation, with the assay calibrated with '25I-labeled DNA under the assumption that each 125I decay forms a double-strand break (10, 11). Low-speed neutral sucrose gradient sedimentation, on the other hand, shows a linear relation between radiation-induced double-strand breaks and dose (12-17). Therefore at least one of these methods must be giving wrong answers, and it is appropriate to discuss the underlying theories for the two approaches.