Paula V. Bennett

Clustered DNA Damages Induced by X Rays in Human Cells

Abstract Sutherland, B. M., Bennett, P. V., Sutherland, J. C. and Laval, J. Clustered DNA Damages Induced by X Rays in Human Cells. Radiat. Res. 157, 611–616 (2002). Although DNA DSBs are known to be important in producing the damaging effects of ionizing radiation in cells, bistranded clustered DNA damages—two or more oxidized bases, abasic sites or strand breaks on opposing DNA strands within a few helical turns—are postulated to be difficult to repair and thus to be critical radiation-induced lesions. Gamma rays can induce clustered damages in DNA in solution, and high-energy iron ions produce DSBs and oxidized pyrimidine clusters in human cells, but it was not known whether sparsely ionizing radiation can produce clustered damages in mammalian cells. We show here that X rays induce abasic clusters, oxidized pyrimidine clusters, and oxidized purine clusters in DNA in human cells. Non-DSB clustered damages comprise about 70% of the complex lesions produced in cells. The relative levels of specific cluster classes depend on the environment of the DNA.

High efficiency detection of bi-stranded abasic clusters in γ-irradiated DNA by putrescine

Bi-stranded abasic clusters, an abasic (AP) site on one DNA strand and another nearby AP site or strand break on the other, have been quantified using Nfo protein from Escherichia coli to produce a double-strand break at cluster sites. Since recent data suggest that Nfo protein cleaves inefficiently at some clusters, we tested whether polyamines, which also cut at AP sites, would cleave abasic clusters at higher efficiency. The data show that Nfo protein cleaves poorly at clusters containing immediately opposed AP sites and those separated by 1 or 3 bp. Putrescine (PUTR) cleaved more efficiently than spermidine or spermine, and did not cleave undamaged DNA. It cleaved abasic clusters in oligonucleotide duplexes more effectively than Nfo protein, including immediately opposed or closely spaced clusters. PUTR cleaved more efficiently than Nfo protein by a factor of approximately 1.7 or approximately 2 for DNA that had been gamma-irradiated in moderate or non-radioquenching conditions, respectively. This suggests that the DNA environment during irradiation affects the spectrum of cluster configurations. Further comparison of PUTR and Nfo protein cleavage may provide useful information on abasic cluster levels and configurations induced by ionizing radiation.

Quantifying DNA damage by gel electrophoresis, electronic imaging and number‐average length analysis

DNA damages that can be converted to single‐ or double strand breaks can be quantified by separating DNA by gel electrophoresis and obtaining a quantitative image of the resulting distribution of DNA in the gel. We review the theory of this method and discuss its implementation, including the charge‐coupled device (CCD) camera systems we developed to acquire images of fluorophore labeled DNA.

Topical glycolic acid enhances photodamage by ultraviolet light

Background: Alpha‐hydroxy acids (AHAs) are widely used as ingredients in cosmetics. Several studies suggest that AHAs can increase the sensitivity of skin to ultraviolet (UV) light.

Low levels of endogenous oxidative damage cluster levels in unirradiated viral and human dnas

Ionizing radiation induces bistranded DNA damage clusters-two or more oxidized bases, abasic, sites or strand breaks on opposing strands within a few helical turns-but it is not known if clusters are also formed in unirradiated DNA in solution or in unirradiated cultured human cells. The frequencies of endogenous oxidized purine clusters (recognized by Escherichia coli Fpg protein), oxidized pyrimidine clusters (recognized by Nth protein), and abasic clusters (cleavage by Nfo protein) were determined using quantitative gel electrophoresis, electronic imaging, and number average length analysis. Methods of DNA isolation and storage were found to affect cluster levels significantly. In bacteriophage T7 DNA prepared using stringent conditions, the frequencies of these clusters were <1/Mbp. In DNA from unirradiated human 28SC monocytes, the levels of such clusters were, at most, a few per gigabase pair.

Proton-HZE-Particle Sequential Dual-Beam Exposures Increase Anchorage-Independent Growth Frequencies in Primary Human Fibroblasts

Abstract Zhou, G., Bennett, P. V., Cutter, N. C. and Sutherland, B. M. Proton-HZE-Particle Sequential Dual-Beam Exposures Increase Anchorage-Independent Growth Frequencies in Primary Human Fibroblasts. Radiat. Res. 166, 488–494 (2006). The radiation field in deep space contains high levels of high-energy protons and substantially lower levels of high-atomic-number, high-energy (HZE) particles. Calculations indicate that cellular nuclei of human space travelers will be hit during a 3-year Mars mission by ∼400 protons and ∼0.4 HZE particles. Thus most cells in astronauts will be hit by a proton(s) before being hit by an HZE particle. To investigate effects of dual ion irradiations on human cells, we irradiated primary human neonatal fibroblasts with protons (1 GeV/nucleon, 20 cGy) followed from 2.5 min to 48 h later by iron or titanium ions (1 GeV/nucleon, 20 cGy) and then measured clonogenic survival and frequency of anchorage-independent growth. This frequency depends on the interval between hydrogen- and iron-ion irradiation, with a critical window between 2.5 min and 1 h producing about three times more anchorage-independent colonies per survivor than expected from simple addition of the two ions separately. The hydrogen-titanium-ion dual-beam irradiation produced similar increases that persisted to ∼6 h. At longer intervals, anchorage-independent growth frequencies were similar to those expected for additivity. However, irradiation of cells with either an iron or a titanium particle first followed by protons produced only additive levels.

DNA damage quantitation by alkaline gel electrophoresis.

Quantifying DNA lesions provides a powerful way to assess the level of endogenous damage or the damage level induced by radiation, chemical or other agents, as well as the ability of cells to repair such damages. Quantitative gel electrophoresis of experimental DNAs along with DNA length standards, imaging the resulting dispersed DNA and calculating the population average length allows accurate measurement of lesion frequencies. Number average length analysis provides high sensitivity and does not require any specific distribution of lesions within the DNA molecules. These methods are readily applicable to strand breaks and ultraviolet radiation induced pyrimidine dimers, but can also be used-with appropriate modifications-for ionizing radiation-induced lesions such as oxidized bases and abasic sites.

Endogenous DNA damage clusters in human hematopoietic stem and progenitor cells.

Clustered DNA damages-multiple oxidized bases, abasic sites, or strand breaks within a few helical turns-are potentially mutagenic and lethal alterations induced by ionizing radiation. Endogenous clusters are found at low frequencies in unirradiated normal human cells and tissues. Radiation-sensitive hematopoietic cells with low glycosylase levels (TK6 and WI-L2-NS) accumulate oxidized base clusters but not abasic clusters, indicating that cellular repair genotype affects endogenous cluster levels. We asked whether other factors, i.e., in the cellular microenvironment, affect endogenous cluster levels and composition in hematopoietic cells. TK6 and WI-L2-NS cells were grown in standard medium (RPMI 1640) alone or supplemented with folate and/or selenium; oxidized base cluster levels were highest in RPMI 1640 and reduced in selenium-supplemented medium. Abasic clusters were low under all conditions. In primary hematopoietic stem and progenitor cells from four non-tobacco-using donors, cluster levels were low. However, in cells from tobacco users, we observed high oxidized base clusters and also abasic clusters, previously observed only in irradiated cells. Protein levels and activity of the abasic endonuclease Ape1 were similar in the tobacco users and nonusers. These data suggest that in highly damaging environments, even normal DNA repair capacity can be overwhelmed, leaving highly repair-resistant clustered damages.

Induction of Anchorage-Independent Growth in Primary Human Cells Exposed to Protons or HZE Ions Separately or in Dual Exposures

Abstract Sutherland, B. M., Cuomo, N. C. and Bennett, P. V. Induction of Anchorage-Independent Growth in Primary Human Cells Exposed to Protons or HZE Ions Separately or in Dual Exposures. Radiat. Res. 164, 493–496 (2005). Travelers on space missions will be exposed to a complex radiation environment that includes protons and heavy charged particles. Since protons are present at much higher levels than are heavy ions, the most likely scenario for cellular radiation exposure will be proton exposure followed by a hit by a heavy ion. Although the effects of individual ion species on human cells are being investigated extensively, little is known about the effects of exposure to both radiation types. One useful measure of mammalian cell damage is induction of the ability to grow in a semi-solid agar medium highly inhibitory to the growth of normal human cells, termed neoplastic transformation. Using primary human cells, we evaluated induction of soft-agar growth and survival of cells exposed to protons only or to heavy charged particles (600 MeV/ nucleon silicon) only as well as of cells exposed to protons followed after a 4-day interval by silicon ions. Both ions alone efficiently transformed the human cells to anchorage-independent growth. Initial experiments indicate that the dose responses for neoplastic transformation of cells exposed to protons and then after 4 days to silicon ions appear similar to that of cells exposed to silicon ions alone.

MELATONIN PROTECTS HUMAN CELLS FROM CLUSTERED DNA DAMAGES, KILLING AND ACQUISITION OF SOFT AGAR GROWTH INDUCED BY X-RAYS OR 970 MEV/N FE IONS

Purpose: We tested the ability of melatonin (N-acetyl-5 methoxytryptamine), a highly effective radical scavenger and human hormone, to protect DNA in solution and in human cells against induction of complex DNA clusters and biological damage induced by low or high linear energy transfer radiation (100 kVp X-rays, 970 MeV/nucleon Fe ions). Materials and methods: Plasmid DNA in solution was treated with increasing concentrations of melatonin (0.0–3.5 mM) and were irradiated with X-rays. Human cells (28SC monocytes) were also irradiated with X-rays and Fe ions with and without 2 mM melatonin. Agarose plugs containing genomic DNA were subjected to Contour Clamped Homogeneous Electrophoretic Field (CHEF) followed by imaging and clustered DNA damages were measured by using Number Average length analysis. Transformation experiments on human primary fibroblast cells using soft agar colony assay were carried out which were irradiated with Fe ions with or without 2 mM melatonin. Results: In plasmid DNA in solution, melatonin reduced the induction of single- and double-strand breaks. Pretreatment of human 28SC cells for 24 h before irradiation with 2 mM melatonin reduced the level of X-ray induced double-strand breaks by ∼50%, of abasic clustered damages about 40%, and of Fe ion-induced double-strand breaks (41% reduction) and abasic clusters (34% reduction). It decreased transformation to soft agar growth of human primary cells by a factor of 10, but reduced killing by Fe ions only by 20–40%. Conclusion: Melatonins effective reduction of radiation-induced critical DNA damages, cell killing, and striking decrease of transformation suggest that it is an excellent candidate as a countermeasure against radiation exposure, including radiation exposure to astronaut crews in space travel.