Jamie R. Milligan

Variation of Single-Strand Break Yield with Scavenger Concentration for Plasmid DNA Irradiated in Aqueous Solution

We have measured the yield of single-strand breaks (SSBs), induced by 137Cs gamma radiation as assayed by agarose gel electrophoresis, for three plasmids and SV40 DNA irradiated in aerobic aqueous solution. DNA SSBs are caused mainly by the hydroxyl radical under these conditions. To characterize the reactivity of DNA with the hydroxyl radical, we investigated the variation of the G value for SSBs [G(SSB)] with the concentration of added hydroxyl radical scavengers. We find that simple competition kinetics does not describe our results, but that a nonhomogeneous kinetics model is in good agreement. At a DNA concentration of 50 micrograms cm-3, G(SSB) for the direct effect is about 1 x 10(-5) mumol J-1 for the DNA substrates studied. This is equivalent to 2 x 10(-10) SSB Gy-1 Da-1. Estimates of the efficiency of SSB induction per OH. radical interaction with DNA (0.32-0.44) reveal that all plasmids are essentially equal in reactivity.

Critical target and dose and dose-rate responses for the induction of chromosomal instability by ionizing radiation

To investigate the critical target, dose response and dose-rate response for the induction of chromosomal instability by ionizing radiation, bromodeoxyuridine (BrdU)-substituted and unsubstituted GM10115 cells were exposed to a range of doses (0.1-10 Gy) and different dose rates (0.092-17.45 Gy min(-1)). The status of chromosomal stability was determined by fluorescence in situ hybridization approximately 20 generations after irradiation in clonal populations derived from single progenitor cells surviving acute exposure. Overall, nearly 700 individual clones representing over 140,000 metaphases were analyzed. In cells unsubstituted with BrdU, a dose response was found, where the probability of observing delayed chromosomal instability in any given clone was 3% per gray of X rays. For cells substituted with 25-66% BrdU, however, a dose response was observed only at low doses (<1.0 Gy); at higher doses (>1.0 Gy), the incidence of chromosomal instability leveled off. There was an increase in the frequency and complexity of chromosomal instability per unit dose compared to cells unsubstituted with BrdU. The frequency of chromosomal instability appeared to saturate around approximately 30%, an effect which occurred at much lower doses in the presence of BrdU. Changing the gamma-ray dose rate by a factor of 190 (0.092 to 17.45 Gy min(-1)) produced no significant differences in the frequency of chromosomal instability. The enhancement of chromosomal instability promoted by the presence of the BrdU argues that DNA comprises at least one of the critical targets important for the induction of this end point of genomic instability.

Yield of single-strand breaks due to attack on DNA by scavenger-derived radicals

We have measured the yield of single-strand breaks (SSBs) in plasmid DNA after 137Cs gamma irradiation, in the presence of dimethyl sulfoxide (DMSO). In the presence of oxygen, the formation of SSBs is due to hydroxyl radical attack. As the DMSO concentration is increased from 10(-4) mol dm-3 to 1 mol dm-3, the SSB yield in the presence and absence of oxygen decreases by over 100-fold and less than 10-fold, respectively. From the DMSO and DNA concentration dependencies of the SSB yield in the absence of oxygen, the second-order rate constant for the reaction of the methyl radical (derived from DMSO) and DNA can be estimated as k2 = 8.8 x 10(4) dm3 mol-1 s-1. Several other scavengers were compared with DMSO under anoxia. Radicals derived from isopropyl alcohol and glycerol also caused SSB formation in DNA, while those from 2-deoxyribose, thymine, 1,3-dimethylthymine and 1,3-dimethyluracil did not. In the case of the scavenger tert-butyl alcohol, it is unclear whether the hydrogen atom (H.) or an organic radical is responsible for the higher SSB yield under anoxic conditions.

Yield of strand breaks as a function of scavenger concentration and LET for SV40 irradiated with 4He ions.

We have measured by gel electrophoresis the yields of single- and double-strand breaks (SSBs and DSBs) induced in aqueous solutions of SV40 DNA and the SV40 minichromosome by 137Cs gamma rays (mean LET 0.3 keV micron-1) and 4He ions (mean LETs 85, 102, and 152 keV microns-1). DNA SSBs are caused mainly by the hydroxyl radicals under these conditions and are reduced in yield as either the hydroxyl radical scavenger concentration or the LET is increased (over the range studied). The G(SSB) for 4He ion irradiation is less by a factor of up to 10 than the G(SSB) for gamma irradiation, depending upon the scavenger concentration. The difference in the yields of SSBs agrees well with the difference in the yields of hydroxyl radicals for the radiations in question. In contrast, the yields of DSBs are similar for gamma and 4He ion irradiation over much of the range of scavenging capacity studied. However, at the highest scavenger concentrations the yields of DSBs are greater for 4He ion irradiation. In addition, the yields of DSBs remain almost constant with increasing LET (over the range studied). Therefore the relative yield of DSBs per SSB increases with increasing LET, supporting the hypothesis that increasing LET leads to an increased clustering of damage in DNA.

Variation of single-strand break yield with scavenger concentration for the SV40 minichromosome irradiated in aqueous solution.

We have measured the yield of single-strand breaks (SSBs) induced in aerobic aqueous solution by 137Cs gamma irradiation for the SV40 minichromosome as measured by agarose gel electrophoresis. Under these conditions, DNA SSBs are caused mainly by the hydroxyl radical. To characterize the reactivity of the SV40 minichromosome with the hydroxyl radical and to compare its behavior with that of naked DNA, we examined the variation of the G value for SSB formation, G(SSB), with the concentration of added hydroxyl radical scavengers. We find that simple competition kinetics is not applicable, but that a nonhomogeneous kinetics model is in much better agreement. Estimates of the efficiency of SSB induction per OH radical interaction with the SV40 minichromosome (0.04-0.05) indicate that this substrate is about five times more radioresistant than naked DNA at scavenging capacities < 10(8) s-1. At a DNA concentration of 50 micrograms ml-1, G(SSB) for the direct effect in the minichromosome is about 1 x 10(-5) mumol J-1 (2 x 10(-10) SSB Gy-1 Da-1), essentially equal to that for naked DNA.

DNA repair by thiols in air shows two radicals make a double-strand break.

Using agarose gel electrophoresis, we have measured the yields of DNA single- and double-strand breaks (SSBs and DSBs) for plasmid DNA gamma-irradiated in aerobic aqueous solution. The presence during irradiation of either of the thiols cysteamine or N-(2-thioethyl)-1,3-diaminopropane (WR-1065) resulted in a concentration-dependent decrease in the yield of SSBs and a much greater decrease in the yield of DSBs. This large differential protective effect was not produced by thioethers or an alcohol of structural similarity to the two thiols, suggesting that repair of DSB radical precursors by thiols is more efficient than for SSB precursors. These observations suggest the existence of a diradical intermediate in the formation of DSBs. The results argue against a major contribution by a single radical mechanism involving interstrand radical transfer via hydrogen abstraction by a peroxyl intermediate, since the half-life of this radical transfer reaction appears to be significantly greater than the lifetime of the intermediate.

The Difference that Linear Energy Transfer Makes to Precursors of DNA Strand Breaks

Using agarose gel electrophoresis, we have measured the yields of DN A single- and double-strand breaks (SSBs and DSBs) for plasmid DNA irradiated in aerobic aqueous solution with either 137Cs gamma rays or 4He ions with a mean LET of 94 or 150 keV micron-1. The presence of dimethyl sulfoxide (DMSO) resulted in a decrease in the yields of both SSBs and DSBs, with a greater decrease being apparent for gamma irradiation than for 4He-ion irradiation. Irradiation by 4He ions in the presence of N-(2-thioethyl)- 1,3-diaminopropane (WR-1065) resulted in a decrease in the yield of SSBs and a slightly larger decrease in the yield of DSBs. Together with results obtained previously, these observations suggest a substantial contribution to the formation of SSBs and DSBs by 4He ions by species containing at least two radicals and more than two radicals, respectively.

On the Chemical Yield of Base Lesions, Strand Breaks, and Clustered Damage Generated in Plasmid DNA by the Direct Effect of X Rays

Abstract Purkayastha, S., Milligan, J. R. and Bernhard, W. A. On the Chemical Yield of Base Lesions, Strand Breaks, and Clustered Damage Generated in Plasmid DNA by the Direct Effect of X Rays. Radiat. Res. 168, 357–366 (2007). The purpose of this study was to determine the yield of DNA base damages, deoxyribose damage, and clustered lesions due to the direct effects of ionizing radiation and to compare these with the yield of DNA trapped radicals measured previously in the same pUC18 plasmid. The plasmids were prepared as films hydrated in the range 2.5 < Γ < 22.5 mol water/mol nucleotide. Single-strand breaks (SSBs) and double-strand breaks (DSBs) were detected by agarose gel electrophoresis. Specific types of base lesions were converted into SSBs and DSBs using the base-excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). The yield of base damage detected by this method displayed a strikingly different dependence on the level of hydration (Γ) compared with that for the yield of DNA trapped radicals; the former decreased by 3.2 times as Γ was varied from 2.5 to 22.5 and the later increased by 2.4 times over the same range. To explain this divergence, we propose that SSB yields produced in plasmid DNA by the direct effect cannot be analyzed properly with a Poisson process that assumes an average of one strand break per plasmid and neglects the possibility of a single track producing multiple SSBs within a plasmid. The yields of DSBs, on the other hand, are consistent with changes in free radical trapping as a function of hydration. Consequently, the composition of these clusters could be quantified. Deoxyribose damage on each of the two opposing strands occurs with a yield of 3.5 ± 0.5 nmol/J for fully hydrated pUC18, comparable to the yield of 4.1 ± 0.9 nmol/J for DSBs derived from opposed damages in which at least one of the sites is a damaged base.

The effect of superhelical density on the yield of single-strand breaks in γ-irradiated plasmid DNA

Using agarose gel electrophoresis, the formation of DNA single-strand breaks (SSBs) by 137Cs gamma irradiation was quantified in negatively supercoiled topological isomers of plasmid pUC18. The G value for SSB formation falls slightly from 1 x 10(8) to 8 x 10(-9) SSB Gy-1 Da-1 as the superhelical density varies from 0.00 to -0.08. This result is not in agreement with recent observations by others which suggest that increasing the negative superhelical density of plasmid DNA increases its sensitivity to X irradiation.

Synthesis and catalytic activity of pluronic stabilized silver–gold bimetallic nanoparticles

In this report, we demonstrate a rapid, simple, and green method for synthesizing silver-gold (Ag-Au) bimetallic nanoparticles (BNPs). We used a novel modification to the galvanic replacement reaction by suspending maltose coated silver nanoparticles (NPs) in ≈ 2% aqueous solution of EO100PO65EO100 (Pluronic F127) prior to HAuCl4 addition. The Pluronic F127 stabilizes the BNPs, imparts biocompatibility, and mitigates the toxicity issues associated with other surfactant stabilizers. BNPs with higher Au:Ag ratios and, subsequently, different morphologies were successfully synthesized by increasing the concentration of gold salt added to the Ag NP seeds. These BNPs have enhanced catalytic activities than typically reported for monometallic Au or Ag NPs (∼ 2-10 fold) of comparable sizes in the sodium borohydride reduction of 4-nitrophenol. The 4-nitrophenol reduction rates were highest for partially hollow BNP morphologies.