Visual quantification of DNA double-strand breaks in bacteria.

In this paper, we describe a method for the visualization of double-strand breaks in a single electrostretched Escherichia coli DNA molecule. We also provide evidence that electrostretched or migrated DNA under neutral microgel electrophoresis conditions is made up of individual chromosomes. Using the neutral microgel electrophoresis technique, DNA migration (stretching) was measured and the number of DNA double-strand breaks were counted following exposure of E. coli cells to 0, 12.5, 25, 50, or 100 rad of X-rays. The use of an intense fluorescent dye, YOYO and custom-made slides have helped us in visualizing individual bacterial DNA molecules. Bacterial DNA appears similar in structure compared to electrostretched DNA from human lymphocytes. We were able to detect changes in DNA migration (stretching) induced by an X-ray dose as low as 12.5 rad and an increase in the number of DNA breaks induced by a dose as low as 25 rad. The extent of DNA migration and number of breaks were directly correlated to X-ray dosage.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on cell killing and interphase chromosome breakage in radiation-sensitive cells

The effect of BrdU incorporation on cell radiosensitivity as well as on the induction of chromosome damage by radiation was studied in plateau-phase xrs-5 cells using the premature chromosome condensation (PCC) method. It is well known that xrs-5 cells are sensitive to ionizing radiation and defective in the repair of radiation-induced DNA double-strand breaks, chromosome damage, and potentially lethal damage (PLD). Compared to repair-proficient CHO 10B cells, a reduction was observed in the overall BrdU-mediated radiosensitization in plateau-phase xrs-5 cells for the same degree of thymidine replacement. This finding is interpreted with a model for BrdU-induced radiosensitization advanced previously, in which two distinct components act to produce the overall radiosensitization observed. One component involves processes associated with the increase in initial damage (DNA and chromosome) production per unit absorbed dose and causes an increase in the slope of the survival curve, while the second component involves enhanced fixation of radiation-induced damage (PLD) and causes a reduction in the width of the shoulder of the survival curve. It is suggested that in plateau-phase xrs-5 cells, the deficiency in the repair of radiation-induced damage compromises BrdU-mediated radiosensitization by leaving active only the radiosensitization component that is associated with an increase in damage induction. Enhancement of cell killing by BrdU in plateau-phase xrs-5 cells resulted in a decrease in D0, the relative value of which was similar to the relative increase in the production of chromosome damage as measured by the PCC method. The relative values for the change in D0 and the production of chromosome aberrations were similar in plateau-phase CHO 10B and xrs-5 cells, suggesting that the physicochemical and/or biochemical processes associated with this phenomenon are the same in the two cell lines. Radiosensitization of a magnitude similar to that observed in exponentially growing CHO 10B cells was induced by BrdU in exponentially growing xrs-5 cells. This effect is attributed to a partial expression of the repair gene (transiently during S phase in all cells, or throughout the cycle in a fraction of cells) that permits some repair of radiation-induced damage and which is compromised by BrdU.     

Radiosensitization by hypoxic pretreatment with misonidazole: an interaction of damage at the DNA level

Prolonged exposures to misonidazole (MISO) in vitro under hypoxic conditions result in radiosensitization which is characterized by a decrease in the size of the radiation survival curve shoulder for cells irradiated under hypoxic or aerobic conditions after drug removal. Although intracellular glutathione (GSH) was depleted during hypoxic exposures to MISO, this could not account for the dose-additive radiosensitization (decrease in shoulder size) since GSH depletion by diethylmaleate had no effect on the sensitivity of cells irradiated in air. The alkaline elution assay was used to measure DNA strand breaks and their repair after exposure to MISO, graded doses of X rays, and the combination of MISO pretreatment with X rays. The elution rate of DNA from irradiated cells increased linearly with X-ray dose, with and without MISO pretreatment. However, the DNA elution rates measured after MISO pretreatment were greater by a constant amount at all X-ray doses greater than 1 Gy. In terms of both cell survival and DNA elution rate, MISO-pretreated cells behaved as though they had received an extra 1.5 Gy. Although the initial damage after X rays was greater in MISO-pretreated cells, there was no effect of MISO pretreatment on the rate of repair of radiation-induced DNA strand breaks. The agreement between the differences in survival levels and DNA elution rates for irradiated control and MISO-pretreated cells and absence of an effect on DNA repair rates suggest that the pretreatment sensitization is due to an additive interaction of damage at the DNA level.     

Radiosensitization and production of DNA double-strand breaks in U87MG brain tumor cells induced by tetraiodothyroacetic acid (tetrac)

We describe the steady-state levels and molecular and cellular repair of DNA double-strand breaks (DSBs) in tetraiodothyroacetic acid (tetrac)-treated human U87MG glioblastoma cells after x-irradiation in vitro. This study was conducted to provide a basis for our previous observation of radiosensitization and inhibition of cellular recovery after irradiation of tetrac-exposed GL261 murine brain tumor cells. We used the neutral comet assay to assess DSBs, and found that the steady-state DSB levels as indicated by the mean tail moment after a 1 h application of 2 nM tetrac at 37oC was increased from a value of 6.1 in control cells to 12.4 in tetrac treated cells at 0 radiation dose. However, at all radiation doses, the induction curves of DSBs were parallel, suggesting that no interaction of tetrac with the initial physical-chemical actions of ionizing radiation occurred. Flow cytometric measurements indicated that this increase was not due to alterations in the relative percentages of U87MG cells throughout the cell cycle. In split-dose DNA repair studies we found that tetrac decreased the repair rate of U87 cells by a factor of 72.5%. This suggests that the radiosensitization from graded single doses of x-rays occurs as a consequence of tetrac inhibition of the post-irradiation repair process. These results link the previously noted changes in cellular endpoints to a molecular endpoint. That is, tetrac produces increased numbers of DSBs in the unirradiated steady-state coupled with a decreased repair rate of DSBs in fractionated radiation experiments.     

Mechanism of radiosensitization by halogenated pyrimidines: bromodeoxyuridine and beta-arabinofuranosyladenine affect similar subsets of radiation-induced potentially lethal lesions in plateau-phase Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells were grown to plateau phase in the presence of various amounts of bromodeoxyuridine (BrdU) and treated after irradiation with beta-arabinofuranosyladenine (ara-A), an inhibitor of DNA and potentially lethal damage (PLD) repair, in order to investigate the importance of repair reactions in general and of PLD repair, in particular, on the mechanism of radiosensitization by halogenated pyrimidines. The degree of BrdU-mediated radiosensitization observed in ara-A-treated cells was compared to that of cells incubated after irradiation in the absence of ara-A. A substantial reduction in BrdU-mediated radiosensitization was observed in cells treated with ara-A at concentrations that, when given alone, produced maximum potentiation in cell killing (500-1500 microM). The residual BrdU-mediated radiosensitization observed at high levels of thymidine replacement could be explained by a BrdU-mediated increase in DNA and chromosome damage induction per gray. These findings are similar to those reported previously for a repair-deficient mutant of CHO cells, the xrs-5 cell line, and consistent with the hypothesis that BrdU-mediated radiosensitization has two distinct components, one that derives from an increase in damage induction per gray, and a second one that derives from an effect of BrdU on the repair of radiation-induced damage. It is proposed that the reduction in BrdU-mediated radiosensitization observed in ara-A-treated cells is the result of ara-A-mediated expression of radiation damage, the repair of which would have been otherwise modulated by BrdU. Since ara-A is known to act by fixing a form of radiation-induced PLD (alpha-PLD), we further propose that BrdU acts by fixing alpha-PLD. A synergistic effect in the potentiation of cell killing was observed between ara-A and BrdU when ara-A was given at concentrations below 100 microM. This result suggests that a benefit may be expected in the clinic from the combined application of halogenated pyrimidines with repair inhibitors, if administered at a carefully screened range of concentrations.     

Detection of ionizing radiation-induced DNA double-strand breaks by filter elution is affected by nuclear chromatin structure

Chinese hamster ovary cells were irradiated with 250 kVp X rays and analyzed for the presence of DNA double-strand breaks using either polycarbonate filter elution or pulsed-field agarose gel electrophoresis at neutral pH. Reduction in DNA length detected by filter elution was produced as a nonlinear function of increasing radiation dose, with a quasi-threshold at low total dose, and as a first-order function of increasing radiation dose as detected by gel electrophoresis. The quasi-threshold observed with filter elution was eliminated when nuclei were isolated from irradiated cells and their chromatin relaxed in a buffer containing low-molarity monovalent cation prior to analysis by filter elution. The results suggest either that the chemical structure of the DNA double-strand breaks produced by low-LET radiation necessitates a DNA relaxation step before they can be detected accurately by filter elution, or that at low total radiation dose a DNA complex forms on the polycarbonate filter.     

应用SCGE技术研究细胞DNA损伤的原理与方法

对单细胞微凝胶电泳（ＳＣＧＥ）技术的操作过程,技术原理以及实验操作过程中应注意的事项,进行了详细介绍和讨论;并应用ＳＣＧＥ技术研究了γ射线照射对人血淋巴细胞ＤＮＡ的损伤效应．结果表明,γ射线照射能引起细胞ＤＮＡ迁移长度增加,且呈显著地剂量效应关系．     

The role of low intracellular or extracellular pH in sensitization to hyperthermic radiosensitization

Previous work showed that intracellular pH (pHi) and not extracellular pH (pHe) was the determinant in the low pH sensitization of hyperthermic killing. The present studies show that the same is true for heat-induced radiosensitization and loss of cellular DNA polymerase activities. Chinese hamster ovary cells after they had adapted to low pH (6.7) had an increase in pHi which rendered cells partially resistant to the low pH sensitization of heat-induced cell killing, radiosensitization, and loss of cellular DNA polymerase activities. These results were quantified by plotting versus pHe, both the thermal enhancement ratio (TER), defined as the ratio of the X-ray dose without heat to the X-ray dose with heat to give an isosurvival value of 0.01, and the thermal enhancement factor (TEF), defined as the ratio of the D0 of the radiation survival curve to the D0 of the radiation survival curve for heat plus radiation. Both the TER and TEF were higher for the unadapted cells than for the adapted cells, i.e., 1.3-1.4 fold higher at a pHe of 6.3. However, the TER or TEF plotted versus pHi was identical for the two cell types. Finally, heat-induced loss of cellular DNA polymerase activities correlated with pHi and not pHe. Therefore, we conclude that pHi and not pHe is responsible for the increase by acid in heat-induced radio-sensitization and loss of cellular DNA polymerase activities.     

Hyperthermic radiosensitization of thermotolerant Chinese hamster ovary cells

Synchronous G1 cells were given a priming dose of heat (45.5 degrees C for 15 min) and then heated and irradiated 6-120 h later. Compared to heat radiosensitization for cells irradiated 10 min after the priming heat dose (thermal enhancement ratio, TER of 2.6 for a 10-fold reduction in survival), heat radiosensitization 18-24 h after the priming heat dose was less (i.e., TER of 1.6 for radiation at 24 h compared with heat-radiation at 24 h). A thermotolerance ratio (TTR) at 24 h was calculated to be 2.6/1.6 = 1.6. TERs at 100-fold or 1000-fold reduction in survival and ratios of slopes of radiation survival curves also showed that the cells developed a similar amount of thermotolerance for heat radiosensitization at 18-24 h. Furthermore, since the TER for heat radiosensitization increased with heat killing either from the priming heat dose or the second heat dose in a similar manner for single or fractionated doses, the TER for nonthermotolerant and thermotolerant cells was the same when related to the heat damage (i.e., amount of killing from heat alone). When the radiation response of cells heated and irradiated 6-120 h after the priming heat dose was compared with the response of cells receiving radiation only, changes in TER as a function of time after the initial priming heat dose were shown to involve: recovery of heat damage interacting with the subsequent radiation dose, thermotolerance for heat radiosensitization, and redistribution of cells surviving the first heat dose into radioresistant phases of the cell cycle. In fact, redistribution resulted in a minimal TER at 72 h for heat-radiation compared with radiation alone, instead of at 24 h where maximal thermotolerance for heat killing was observed [P. K. Holahan and W. C. Dewey, Radiat. Res. 106, 111 (1986)]. These observations are discussed relative to clinical considerations and similar results reported from in vivo experiments.     

Assessment of radiation-induced DNA damage in human blood lymphocytes using the single-cell gel electrophoresis technique.

The ability of the alkaline single-cell gel (SCG) electrophoresis technique to detect single-strand breaks and alkali-labile DNA damage in human cells induced by low doses of radiation was evaluated. Peripheral blood lymphocytes were irradiated with gamma-rays from a 137Cs source at doses from 0.01 to 1 Gy and exposed to alkali (pH greater than 13) for 20, 40 or 60 min and then electrophoresed at 25 V and 300 mA for either 20 or 40 min. The extent of DNA damage that was expressed and detected as DNA migration depended directly on the dose of radiation, the duration of exposure to alkali and the length of electrophoresis. At all experimental conditions tested, it was possible to detect a significant increase in DNA damage induced by a radiation dose as low as 0.05 Gy. Based on an analysis of the ratio of the range to the standard deviation for each radiation dose and experimental condition, the distribution of damage among cells for all doses was neither excessively homogeneous nor heterogeneous. Furthermore, the distribution was independent of radiation treatment. The SCG technique is rapid and sensitive, and useful for investigations concerned with effects of low doses of radiation.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on repair of DNA breaks, interphase chromatin breaks, and potentially lethal damage in plateau-phase CHO cells.

There is evidence suggesting that radiosensitization induced in mammalian cells by substitution in the DNA of thymidine with BrdU has a component that relies on inhibition of repair and/or fixation of radiation damage. Here, experiments designed to study the mechanism of this phenomenon are described. The effect of BrdU incorporation into DNA was studied on cellular repair capability, rejoining of interphase chromosome breaks, as well as induction and rejoining of DNA double- and single-stranded breaks (DSBs and SSBs) in plateau-phase CHO cells exposed to X rays. Repair of potentially lethal damage (PLD), as measured by delayed plating of plateau-phase cells, was used to assay cellular repair capacity. Rejoining of interphase chromosome breaks was assayed by means of premature chromosome condensation (PCC); induction and rejoining of DNA DSBs were assayed by pulsed-field gel electrophoresis and induction and rejoining of DNA SSBs by DNA unwinding. A decrease was observed in the rate of repair of PLD in cells grown in the presence of BrdU, the magnitude of which depended upon the degree of thymidine replacement. The relative increase in survival caused by PLD repair was larger in cells substituted with BrdU and led to a partial loss of the radiosensitizing effect compared to cells tested immediately after irradiation. A decrease was also observed in the rate of rejoining of interphase chromosome breaks as well as in the rate of rejoining of the slow component of DNA DSBs in cells substituted with BrdU. The time constants measured for the rejoining of the slow component of DNA DSBs and of interphase chromosome breaks were similar both in the presence and in the absence of BrdU, suggesting a correlation between this subset of DNA lesions and interphase chromosome breaks. It is proposed that a larger proportion of radiation-induced potentially lethal lesions becomes lethal in cells grown in the presence of BrdU. Potentially lethal lesions are fixed via interaction with processes associated with cell cycle progression in cells plated immediately after irradiation, but can be partly repaired in cells kept in the plateau-phase. It is hypothesized that fixation of PLD is caused by alterations in chromatin conformation that occur during normal progression of cells throughout the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of ionizing radiation on DNA synthesis in ataxia telangiectasia cells.

The effect of ionizing radiation on DNA synthesis in control and ataxia telangiectasia (AT) lymphoblastoid cell lines was determined. A dose dependent decrease in DNA synthesis was observed in control cells, and the rate and extent of thi decrease in synthesis increased with time after irradiation. No decrease in DNA synthesis was obtained in AT cells, immediately following irradiation, at doses up to 400 rads. At longer times postirradiation, inhibition of synthesis increased but the extent of inhibition was less in AT cell than controls at all doses used. An immediate depression of DNA synthesis was evident in control cells after a radiation dose of 200 rads reaching a maximum at 90 min postirradiation. Little or no decrease in DNA synthesis was evident in AT cells up to 60 min after the same radiation dose, but a decrease occurred between 60 and 90 min after irradiation. The rate of recovery of DNA synthesis to normal levels was more rapid in AT cells than in controls.     

Radiosensitization by cisplatin of RIF1 tumour cells in vitro

The ability of cis-diamminedichloroplatinum (II) (c-DDP) to enhance radiation-induced cell killing was tested on oxic RIF1 tumour cells in monolayer culture. Marked radiosensitization of the survivors of a 1 h drug treatment was found with all c-DDP doses tested, with enhancement ratios increasing from 1.2 to 2.2 with increasing drug dose. Isobologram analyses showed that the interactions of c-DDP with X-rays were supra-additive. To test whether part of the enhancement was due to a selection of subpopulations, the diploid and tetraploid RIF1 cells, which normally coexist in culture, were separated by unit gravity velocity sedimentation, and by developing diploid and tetraploid clones. Both methods showed that there was little difference in either drug sensitivity or radiation sensitivity between diploid and tetraploid cells. DNA histograms obtained by flow cytometry showed little or no cycle progression during the 1 h drug treatment. These data indicate that the radiosensitization was not the result of the drug exposure leaving cells in a radiosensitive phase. The observed radiosensitization, therefore, appears to have resulted from a true drug/X-ray interaction.     

Electrophoresis of DNA in oriented agarose gels

Oriented agarose gels were prepared by applying an electric field to molten agarose while it was solidifying. Immediately afterwards, DNA samples were applied to the gel and electrophoresed in a constant unidirectional electric field. Regardless of whether the orienting field was applied parallel or perpendicular to the eventual direction of electrophoresis, the mobilities of linear and supercoiled DNA molecules were either faster (80% of the time) or slower (20% of the time) than observed in control, unoriented gels run simultaneously. The difference in mobility in the oriented gel (whether faster or slower) usually increased with increasing DNA molecular weight and increasing voltage applied to orient the agarose matrix. In perpendicularly oriented gels linear DNA fragments traveled in lanes skewed toward the side of the gel; supercoiled DNA molecules traveled in straight lanes. If the orienting voltage was applied parallel to the direction of electrophoresis, both linear and supercoiled DNA molecules migrated in straight lanes. These effects were observed in gels cast from different types of agarose, using various agarose concentrations and two different running buffers, and were observed both with and without ethidium bromide incorporated in the gel. Similar results were observed if the agarose was allowed to solidify first, and the orienting electric field was then applied to the gel for several hours before the DNA samples were added and electrophoresed. The results suggest that the agarose matrix can be oriented by electric fields applied to the gel before and probably during electrophoresis, and that orientation of the matrix affects the mobility and direction of migration of DNA molecules. The skewed lanes observed in the perpendicularly oriented gels suggest that pores or channels can be created in the matrix by application of an electric field. The oriented matrix becomes randomized with time, because DNA fragments in oriented and unoriented gels migrated in straight lanes with identical velocities 24 hours later.     

Dominant-negative cAMP-responsive element-binding protein inhibits proliferating cell nuclear antigen and DNA repair,leading to increased cellular radiosensitivity

Selective inhibition of the epidermal growth factor receptor or mitogen-activated protein kinase (MAPK) results in radiosensitization of cancer cells. One potential mechanism involves cAMP-responsive element-binding protein, which is activated by radiation via the epidermal growth factor receptor/MAPK pathway and which regulates synthesis of proliferating cell nuclear antigen (PCNA), a protein involved in repair of ionizing radiation-induced DNA damage. To test for a role of CREB in cellular radiosensitivity, CHO cells were transfected with plasmids expressing dominant-negative CREB mutants (CR133 or KCREB), and various end-points were measured 48 h later. Basal levels of PCNA-CAT reporter construct activity were reduced by 60 and 40% following expression of CR133 and KCREB, respectively; similar decreases were observed in PCNA protein levels. Pulsed-field gel electrophoresis measurements showed that CR133 inhibited the repair of radiation-induced DNA double-strand breaks, and this effect was reversed by over-expression of PCNA; dominant-negative CREB also significantly inhibited split-dose recovery. Clonogenic assays were used to determine surviving fraction; the dose enhancement ratios for dominant-negative CREB-expressing cells compared with control (vector alone) were 1.5 and 1.3 for CR133 and KCREB, respectively. Importantly, co-transfection of mutant CREB and a construct constitutively expressing PCNA protein restored radiosensitivity of CHO cells back to wild-type levels. Moreover, cells expressing either CREB mutant showed no significant cell cycle redistribution. These data demonstrate that genetic disruption of CREB results in radiosensitization, and that this effect can be explained by a mechanism involving decreased PCNA expression and inhibition of DNA repair.     

Ames实验及彗星实验检测辅酶NADH的抗突变作用

采用Ames实验及单细胞凝胶电泳（SCGE,彗星实验）,对还原型辅酶NADH进行抗突变研究。NADH中、高剂量组在加或不加S9的情况下,均能不同程度地抑制由致突变物引起的TA98、TA100回变菌落数的增加,降低SCGE拖尾细胞率。表明还原型辅酶NADH具有一定的抗突变作用。     

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.     

Acceleration of DNA-double strand rejoining during the adaptive response of Chlamydomonas reinhardtii

Newly developed constant-field low voltage electrophoresis (adapted for algae cells by us) was applied to quantify the induction and repair of nuclear DNA double-strand breaks, by measuring the movement of DNA out of the starting wells into the electrophoresis gel using a UV-gel scan and computer analysis of DNA-ethidium bromide fluorescense (Syngene; Gene tools). A cell-wall-less mutant strain of Chlamydomonas reinhardtii (CW15) was used; the DNA and proteins are easily accessible because of the lack of an outer cell wall. Our results showed that giving a small priming dose (50 Gy) led to a small acceleration of dsb rejoining. When the magnitude of the priming dose was progressively increased, there was a corresponding decrease in the fraction of damage remaining at 4 hours after radiation exposure (to a test dose of 500 Gy). This indicates an upregulated rejoining of dsb following exposure of cells to the priming dose, which may be related to the strong adaptive response in this organism. Protein synthesis inhibitors were found to reduce the rate of rejoining of dsb, and from earlier results are known to inhibit the adaptive response. Thus, the adaptive response is likely to be dependent on increased dsb rejoining and depends on de novo protein synthesis. The nature of these proteins has not yet been established. C. reinhardtii CW15 is an attractive model system in which to study the underlying mechanisms of the adaptive response to ionizing radiation, and its underlying link with dsb rejoining. The results are interesting both from a basic biological point of view, and as a means to further understand the response of tumour cells to radiation therapy since the adaptive response has been postulated to determine the shape of the "shoulder" region of the survival curve of cells at low doses of radiation.     

DNA interduplex crosslinks induced by Al(Kalpha) X rays under vacuum

Dry pGEM-3Zf(-) plasmid DNA was exposed to Al(kalpha) X rays (1.5 keV) for various times in an ultra-high vacuum chamber with mean absorbed dose rates ranging from 1.8 to 41.7 Gy s(-1). The different forms of plasmid DNA were separated by neutral agarose gel electrophoresis and quantified by staining and laser scanning. In addition to the bands for supercoiled, nicked circular, linear and concatameric forms of plasmid DNA, two additional bands were observed in X-irradiated samples; these migrated at rates similar to those for 8-kb and >10-kb linear double-stranded DNA. Digestion of irradiated DNA with the restriction enzymes EcoR1 and PvuI suggested that the two slowly migrating bands were interduplex crosslinked DNA. Alkaline agarose gel electrophoresis of irradiated DNA digested with EcoR1 confirmed that the interduplex crosslink was covalent. Exposure-response curves were determined for the formation of nicked circular, linear and interduplex crosslinked DNA as well as for the loss of supercoiled and concatameric DNA. Formation and loss of these species were independent of absorbed dose rate over a 20-fold range. The G values for DNA single-strand breaks, double-strand breaks and crosslinks were determined to be 62 +/- 6, 5.6 +/- 0.6 and 16 +/- 4 nmol J(-1), respectively. The formation of DNA interduplex crosslinks appears to be due to single event. The mechanism responsible for the formation of DNA interduplex crosslinks is discussed with emphasis on its implications in vivo.     

Voltammetric determination of gamma radiation-induced DNA damage

Homopolydeoxyribonucleotides, poly[dGuo], poly[dAdo], poly[dThd], and poly[dCyd], and calf thymus single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) aqueous solutions previously exposed to gamma radiation doses between 2 and 35 Gy, were studied by differential pulse voltammetry using a glassy carbon electrode. The interpretation of the voltammetric data was also supported by the electrophoretic migration profile obtained for the same ssDNA and dsDNA gamma-irradiated samples by nondenaturing agarose gel electrophoresis. The generation of 8-oxo-7,8-dihydroguanine, 2,8-dihydroxyadenine, 5-formyluracil, base-free sites, and single- and double-stranded breaks in the gamma-irradiated DNA samples was detected voltammetrically, with the amount depending on the irradiation time. It was found that the current peaks obtained for 8-oxoguanine increase linearly with the radiation dose applied to the nucleic acid sample, and values between 8 and 446 8-oxo-7,8-dihydroguanine (8-oxoGua) per 10(6) guanines per Gy were obtained according to the nucleic acid sample. The results showed that voltammetry can be used for monitoring and simultaneously characterizing different kinds of DNA damage caused by gamma radiation exposure.