Darel J. Hunting

Radiosensitization of DNA by Gold Nanoparticles Irradiated with High-Energy Electrons

Abstract Zheng, Y., Hunting, D. J., Ayotte, P. and Sanche, L. Radiosensitization of DNA by Gold Nanoparticles Irradiated with High-Energy Electrons. Radiat. Res. 168, 19–27 (2008). Thin films of pGEM-3Zf(−) plasmid DNA were bombarded by 60 keV electrons with and without gold nanoparticles. DNA single- and double-strand breaks (SSBs and DSBs) were measured by agarose gel electrophoresis. From transmission electron micrographs, the gold nanoparticles were found to be closely linked to DNA scaffolds, probably as a result of electrostatic binding. The probabilities for formation of SSBs and DSBs from exposure of 1:1 and 2:1 gold nanoparticle:plasmid mixtures to fast electrons increase by a factor of about 2.5 compared to neat DNA samples. For monolayer DNA adsorbed on a thick gold substrate, the damage increases by an order of magnitude. The results suggest that the enhancement of radiosensitivity is due to the production of additional low-energy secondary electrons caused by the increased absorption of ionizing radiation energy by the metal, in the form of gold nanoparticles or of a thick gold substrate. Since short-range low-energy secondary electrons are produced in large amounts by any type of ionizing radiation, and since on average only one gold nanoparticle per DNA molecule is needed to increase damage considerably, targeting the DNA of cancer cells with gold nanoparticles may offer a novel approach that is generally applicable to radiotherapy treatments.

Cross Sections for Low-Energy (10 – 50 eV) Electron Damage to DNA

Abstract Boudaïffa, B., Cloutier, P., Hunting, D., Huels, M. A. and Sanche, L. Cross Sections for Low-Energy (10 – 50 eV) Electron Damage to DNA. Radiat. Res. 157, 227 – 234 (2002). We report direct measurements of the formation of single-, double- and multiple strand breaks in pure plasmid DNA as a function of exposure to 10 – 50 eV electrons. The effective cross sections to produce these different types of DNA strand breaks were determined and were found to range from approximately 10−17 to 3  × 10−15 cm2. The total effective cross section and the effective range for destruction of supercoiled DNA extend from 3.4 to 4.4  × 10−15 cm2 and 12 to 14 nm, respectively, over the range 10 – 50 eV. The variation of the effective cross sections with electron energy is discussed in terms of the electrons inelastic mean free path, penetration depth, and dissociation mechanisms, including resonant electron capture; the latter is found to dominate the effective cross sections for single- and double-strand breaks at 10 eV. The most striking observations are that (1) supercoiled DNA is approximately one order of magnitude more sensitive to the formation of double-strand breaks by low-energy electrons than is relaxed circular DNA, and (2) the dependence of the effective cross sections on the incident electron energy is unrelated to the corresponding ionization cross sections. This finding suggests that the traditional notion that radiobiological damage is related to the number of ionization events would not apply at very low energies.

Effective Cross Sections for Production of Single-Strand Breaks in Plasmid DNA by 0.1 to 4.7 eV Electrons

Abstract Panajotovic, R., Martin, F., Cloutier, P., Hunting, D. and Sanche, L. Effective Cross Sections for Production of Single-Strand Breaks in Plasmid DNA by 0.1 to 4.7 eV Electrons. Radiat. Res. 165, 452–459 (2006). We determined effective cross sections for production of single-strand breaks (SSBs) in plasmid DNA [pGEM 3Zf(-)] by electrons of 10 eV and energies between 0.1 and 4.7 eV. After purification and lyophilization on a chemically clean tantalum foil, dry plasmid DNA samples were transferred into a high-vacuum chamber and bombarded by a monoenergetic electron beam. The amount of the circular relaxed DNA in the samples was separated from undamaged molecules and quantified using agarose gel electrophoresis. The effective cross sections were derived from the slope of the yield as a function of exposure and had values in the range of 10−15– 10−14 cm2, giving an effective cross section of the order of 10−18 cm2 per nucleotide. Their strong variation with incident electron energy and the resonant enhancement at 1 eV suggest that considerable damage is inflicted by very low-energy electrons to DNA, and it indicates the important role of π* shape resonances in the bond-breaking process. Furthermore, the fact that the energy threshold for SSB production is practically zero implies that the sensitivity of DNA to electron impact is universal and is not limited to any particular energy range.

Single-Strand-Specific Radiosensitization of DNA by Bromodeoxyuridine

Abstract Cecchini, S., Girouard, S., Huels, M. A., Sanche, L. and Hunting, D. J. Single-Strand-Specific Radiosensitization of DNA by Bromodeoxyuridine. Radiat. Res. 162, 604–615 (2004). The effects of bromodeoxyuridine (BrdUrd) substitution for thymidine on γ-ray-induced strand breakage were determined in single- and double-stranded oligonucleotides and double-stranded oligonucleotides containing a mismatched bubble region. BrdUrd does not sensitize complementary double-stranded DNA to γ-ray-induced strand breakage, but it greatly sensitizes single-stranded DNA. However, when the BrdUrd is present in a single-stranded bubble of a double-stranded oligonucleotide, the non-base-paired nucleotides adjacent to the BrdUrd as well as several unpaired sites on the opposite unsubstituted strand are strongly sensitized. The radiosensitization properties of BrdUrd result primarily from the electrophilic nature of the bromine, making it a good leaving group and leading to the irreversible formation of the uridine-yl radical (dUrd·) or the uridine-yl anion (dUrd−) upon addition of an electron. The radiolytic loss of the bromine atom is greatly suppressed in double-stranded compared to single-stranded DNA. Thus we propose that the radiosensitization effects of bromouracil in vivo will likely be limited to single-strand regions such as found in transcription bubbles, replication forks, DNA bulges and the loop region of telomeres. Our results may have profound implications for the clinical use of bromodeoxyuridine (BrdUrd) as a radiosensitizer as well as for the development of targeted radiosensitizers.

Phosphodiester and N-glycosidic bond cleavage in DNA induced by 4-15 eV electrons

Thin molecular films of the short single strand of DNA, GCAT, were bombarded under vacuum by electrons with energies between 4 and 15 eV. Ex vacuo analysis by high-pressure liquid chromatography of the samples exposed to the electron beam revealed the formation of a multitude of products. Among these, 12 fragments of GCAT were identified by comparison with reference compounds and their yields were measured as a function of electron energy. For all energies, scission of the backbone gave nonmodified fragments containing a terminal phosphate, with negligible amounts of fragments without the phosphate group. This indicates that phosphodiester bond cleavage by 4-15 eV electrons involves cleavage of the C-O bond rather than the P-O bond. The yield functions exhibit maxima at 6 and 10-12 eV, which are interpreted as due to the formation of transient anions leading to fragmentation. Below 15 eV, these resonances dominate bond dissociation processes. All four nonmodified bases are released from the tetramer, by cleavage of the N-glycosidic bond, which occurs principally via the formation of core-excited resonances located around 6 and 10 eV. The formation of the other nonmodified products leading to cleavage of the phosphodiester bond is suggested to occur principally via two different mechanisms: (1) the formation of a core-excited resonance on the phosphate unit followed by dissociation of the transient anion and (2) dissociation of the CO bond of the phosphate group formed by resonance electron transfer from the bases. In each case, phosphodiester bond cleavage leads chiefly to the formation of stable phosphate anions and sugar radicals with minimal amounts of alkoxyl anions and phosphoryl radicals.

On the role of low-energy electrons in the radiosensitization of DNA by gold nanoparticles

Four different gold nanoparticle (GNP) preparations, including naked GNPs and GNPs coated either with thiolated undecane (S-C(11)H(23)), or with dithiolated diethylenetriaminepentaacetic (DTDTPA) or gadolinium (Gd) DTDTPA chelating agents, were synthesized. The average diameters, for each type of nanoparticle, are 5 nm, 10 and 13 nm, respectively. Dry films of plasmid DNA pGEM-3Zf(-), DNA with bound GNPs and DNA with coated GNPs were bombarded with 60 keV electrons. The yields of single and double strand breaks were measured as a function of exposure by electrophoresis. The binding of just one GNP without coating to DNA containing 3197 base pairs increases single and double strand breaks by a factor of 2.3 while for GNPs coated with S-C(11)H(23) this factor is reduced to 1.6. The GNPs coated with DTDTPA and DTDTPA:Gd in the same ratio with the DNA, produce essentially no increment in damage. These results could be explained by the attenuation by the coatings of the intensity of the low-energy photoelectrons emitted from the GNPs. Thus, coatings of GNPs may considerably attenuate the short-range low-energy electrons emitted from gold, leading to a considerable decrease of radiosensitization. According to our results, the highest radiosensitization should be obtained with GNPs having the shortest possible ligand, directed to the DNA of cancer cells.

Cisplatin Enhances the Formation of DNA Single- and Double-Strand Breaks by Hydrated Electrons and Hydroxyl Radicals

The synergistic interaction of cisplatin with ionizing radiation is the clinical rationale for the treatment of several cancers including head and neck, cervical and lung cancer. The underlying molecular mechanism of the synergy has not yet been identified, although both DNA damage and repair processes are likely involved. Here, we investigate the indirect effect of γ rays on strand break formation in a supercoiled plasmid DNA (pGEM–3Zf-) covalently modified by cisplatin. The yields of single- and double-strand breaks were determined by irradiation of DNA and cisplatin/DNA samples with 60Co γ rays under four different scavenging conditions to examine the involvement of hydrated electrons and hydroxyl radicals in inducing the DNA damage. At 5 mM tris in an N2 atmosphere, the presence of an average of two cisplatins per plasmid increased the yields of single- and double-strand breaks by factors of 1.9 and 2.2, respectively, relative to the irradiated unmodified DNA samples. Given that each plasmid of 3,200 base pairs contained an average of two cisplatins, this represents an increase in radiosensitivity of 3,200-fold on a per base pair basis. When hydrated electrons were scavenged by saturating the samples with N2O, these enhancement factors decreased to 1.5 and 1.2, respectively, for single- and double-strand breaks. When hydroxyl radicals were scavenged using 200 mM tris, the respective enhancement factors were 1.2 and 1.6 for single- and double-strand breaks, respectively. Furthermore, no enhancement in DNA damage by cisplatin was observed after scavenging both hydroxyl radicals and hydrated electrons. These findings show that hydrated electrons can induce both single- and double-strand breaks in the platinated DNA, but not in unmodified DNA. In addition, cisplatin modification is clearly an extremely efficient means of increasing the formation of both single- and double-strand breaks by the hydrated electrons and hydroxyl radicals created by ionizing radiation.

Transcription-dependent and independent DNA excision repair pathways in human cells

alpha-Amanitin, an inhibitor of RNA polymerase II, has little effect on either UV-induced incision or repair synthesis in cultured normal human fibroblasts but almost completely inhibits both processes in xeroderma pigmentosum group C fibroblasts. Cycloheximide, at a concentration which inhibits protein synthesis by 75-80%, has no effect on incision or repair synthesis in either cell type, which argues that the effects of alpha-amanitin on repair occur at the level of transcription. Cot analysis demonstrates that UV-induced repair synthesis occurs at similar levels in highly repetitive, middle repetitive and single copy sequence in both normal and xeroderma group C cells. We conclude that normal cells must have at least two excision repair pathways for repair of UV-induced damage, one dependent on transcription and the other independent.

DNA interstrand cross-links induced by ionizing radiation: an unsung lesion.

The induction of DNA interstrand cross-links by ionizing radiation has been largely ignored in favour of studies on double-strand break formation and repair. At least part of the problem is technical; it is difficult to detect and quantify interstrand cross-links when the same agent forms both cross-links and single strand breaks because the detection of interstrand cross-links generally involves a denaturation step. Our group has studied the induction of interstrand cross-links following irradiation of DNA containing bromouracil at specific sites. We found that the formation of interstrand cross-links requires the presence of a few (3-5) mismatched bases, comprising the bromouracil. In the absence of mismatched bases, no radiation-induced cross-linking was observed; however, even in the absence of bromouracil, cross-linking still occurred, albeit at a lower efficiency. Our molecular modelling studies demonstrate that the mobility of the bases in the mismatched region is essential for the cross-linking process. Thus, our hypothesis is that ionizing radiation induces DNA interstrand cross-links in non-hybridized regions of DNA. Some obvious examples of such DNA regions are replication forks, transcription bubbles and the D-loop of telomeres. However, an abundance of studies have made it clear that there must be many single-stranded regions in the genome, such as hairpins and cruciforms. For example, alpha satellite DNA, in centromere regions of human chromosomes, forms hairpins. Thus, a variety of non-B DNA structures (hairpins, slipped DNA and tetrahelical structures) exist in the genome and should be susceptible to the formation of radiation-induced interstrand cross-links. Although interstrand cross-links have thus far been virtually ignored in radiation biology, it will be worthwhile to develop methods to detect their presence following exposure of cells to biologically relevant levels of ionizing radiation, since, on a per lesions basis, they are probably more toxic than double-strand breaks.

DNA damage and repair following treatment of V-79 cells with sulfonated phthalocyanines.

Abstract. Two gallium‐phthalocyanines were tested for their effects on cell survival, trypsinization, and DNA strand breaks in intact and permeable cells, measured by alkaline elution. Gallium‐tetrasulfophthalocyanine was not phototoxic to cells and caused no measurable DNA damage in intact cells, while a mixture of less sulfonated gallium‐sulfophthalocyanines, containing an average of 2.7 sulfonates per molecule, was very phototoxic and induced DNA strand breaks and/or alkali‐sensitive sites; however, both drugs were equally effective at inducing DNA strand breaks in permeable cells. The DNA damage was rapidly repaired in intact cells; this process was not inhibited by aphidicolin, an inhibitor of DNA polymerases alpha and delta, under conditions in which DNA replicative synthesis was inhibited by more than 90%.