Response of primary human fibroblasts exposed to solar particle event protons.

Solar particle events (SPEs) present a major radiation-related risk for manned exploratory missions in deep space. Within a short period the astronauts may absorb doses that engender acute effects, in addition to the risk of late effects, such as the induction of cancer. Using primary human cells, we studied clonogenic survival and the induction of neoplastic transformation after exposure to a worst case scenario SPE. We simulated such an SPE with monoenergetic protons (50, 100, 1000 MeV) delivered at a dose rate of 1.65 cGy min⁻¹ in a dose range from 0 to 3 Gy. For comparison, we exposed the cells to a high dose rate of 33.3 cGy min⁻¹. X rays (100 kVp, 8 mA, 1.7 mm Al filter) were used as a reference radiation. Overall, we observed a significant sparing effect of the SPE dose rate on cell survival. High-dose-rate protons were also more efficient in induction of transformation in the dose range below 30 cGy. However, as dose accumulated at high dose rate, the transformation levels declined, while at the SPE dose rate, the number of transformants continued to increase up to about 1 Gy. These findings suggest that considering dose-rate effects may be important in evaluating the biological effects of exposure to space radiation. Our analyses of the data based on particle fluence showed that lethality and transforming potential per particle clearly increased with increasing linear energy transfer (LET) and thus with the decreasing energy of protons. Further, we found that the biological response was determined not only by LET but also type of radiation, e.g. particles and photons. This suggests that using γ or X rays may not be ideal for assessing risk associated with SPE exposures.

Processing of abasic DNA clusters in hApeI-silenced primary fibroblasts exposed to low doses of X-irradiation.

Clustered damage in DNA includes two or more closely spaced oxidized bases, strand breaks or abasic sites that are induced by high- or low-linear-energy-transfer (LET) radiation, and these have been found to be repair-resistant and potentially mutagenic. In the present study we found that abasic clustered damages are also induced in primary human fibroblast cells by low-LET X-rays even at very low doses. In response to the induction of the abasic sites, primary fibroblasts irradiated by low doses of X-rays in the range 10-100 cGy showed dose-dependent up-regulation of the DNA repair enzyme, ApeI. We found that the abasic clusters in primary fibroblasts were more lethal to cells when hApeI enzyme expression was down-regulated by transfecting primary fibroblasts with hApeI siRNA as determined by clonogenic survival assay. Endonuclease activity of hApeI was found to be directly proportional to hApeI gene-silencing efficiency. The DNA repair profile showed that processing of abasic clusters was delayed in hApeI-siRNA-silenced fibroblasts, which challenges the survival of the cells even at very low doses of X-rays. Thus, the present study is the first to attempt to understand the induction of cluster DNA damage at very low doses of low- LET radiation in primary human fibroblasts and their processing by DNA repair enzyme ApeI and their relation with the survival of the cells.

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: Melatonin's 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.

Yields of clustered DNA damage induced by charged-particle radiations of similar kinetic energy per nucleon: LET dependence in different DNA microenvironments.

To determine the linear energy transfer (LET) dependence of the biological effects of densely ionizing radiation in relation to changes in the ionization density along the track, we measured the yields and spectrum of clustered DNA damages induced by charged particles of different atomic number but similar kinetic energy per nucleon in different DNA microenvironments. Yeast DNA embedded in agarose in solutions of different free radical scavenging capacity was irradiated with 1 GeV protons, 1 GeV/nucleon oxygen ions, 980 MeV/nucleon titanium ions or 968 MeV/nucleon iron ions. The frequencies of double-strand breaks (DSBs), abasic sites and oxypurine clusters were quantified. The total DNA damage yields per absorbed dose induced in non-radioquenching solution decreased with LET, with minor variations in radioquenching conditions being detected. However, the total damage yields per particle fluence increased with LET in both conditions, indicating a higher efficiency per particle to induce clustered DNA damages. The yields of DSBs and non-DSB clusters as well as the damage spectra varied with LET and DNA milieu, suggesting the involvement of more than one mechanism in the formation of the different types of clustered damages.

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.

Human abasic endonuclease action on multilesion abasic clusters: implications for radiation-induced biological damage.

Clustered damages-two or more closely opposed abasic sites, oxidized bases or strand breaks-are induced in DNA by ionizing radiation and by some radiomimetic drugs. They are potentially mutagenic or lethal. High complexity, multilesion clusters (three or more lesions) are hypothesized as repair-resistant and responsible for the greater biological damage induced by high linear energy transfer radiation (e.g. charged particles) than by low linear energy transfer X- or gamma-rays. We tested this hypothesis by assessing human abasic endonuclease Ape1 activity on two- and multiple-lesion abasic clusters. We constructed cluster-containing oligonucleotides using a central variable cassette with abasic site(s) at specific locations, and 5' and 3' terminal segments tagged with visually distinctive fluorophores. The results indicate that in two- or multiple-lesion clusters, the spatial arrangement of uni-sided positive [in which the opposing strand lesion(s) is 3' to the base opposite the reference lesion)] or negative polarity [opposing strand lesion(s) 5' to the base opposite the reference lesion] abasic clusters is key in determining Ape1 cleavage efficiency. However, no bipolar clusters (minimally three-lesions) were good Ape1 substrates. The data suggest an underlying molecular mechanism for the higher levels of biological damage associated with agents producing complex clusters: the induction of highly repair-resistant bipolar clusters.

Split-dose exposures versus dual ion exposure in human cell neoplastic transformation.

Since radiation fields of space contain many-fold more protons than high atomic number, high energy (HZE) particles, cells in astronaut crews will experience on average several proton hits before an HZE hit. Thus radiation regimes of proton exposure before HZE particle exposure simulate space radiation exposure, and measurement of the frequency of neoplastic transformation of human primary cells to anchorage-independent growth simulates an initial step in cancer induction. Although previous investigations indicated a synergistic increase in transformation yields in the cells exposed to protons followed by HZE particles, these experiments did not differentiate between the effect of splitting of the dose into two fractions and that of changing the ion beams. To test this, we irradiated cells with split doses of either protons or HZE particles, then measured clonogenic survival and neoplastic transformation, as measured by colony formation in semi-solid soft agar medium. The data show that the split dose of 20 cGy plus 20 cGy of either H or HZE ions gave about the same effect as the 40 cGy uninterrupted dose, quite different from the effect of the mixed ion beam H + HZE irradiation. We also asked if lower proton doses than 20 cGy followed 15 min later by 20 cGy of HZE ions gave greater than additive transformation frequencies. Substantial increases in transformation levels were observed for all proton doses tested, including 1 cGy. These results point to the signal importance of protons in affecting the effect of space radiation on human cells.

Proton-HZE-particle sequential dual-beam exposures increase anchorage-independent growth frequencies in primary human fibroblasts.

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 approximately 400 protons and approximately 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 approximately 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.

Expression of the E. coli fpg protein in CHO cells lowers endogenous oxypurine clustered damage levels and decreases accumulation of endogenous Hprt mutations.

Endogenous DNA damage clusters--two or more oxidized bases, abasic sites, or strand breaks within about 20 base pairs on opposing strands--can accumulate in unirradiated mammalian cells, and may be significant origins of spontaneous detrimental biological effects. Factors determining the levels of such endogenous clusters are largely unknown. To determine if cellular repair genotype can affect endogenous cluster levels in mammalian cells, the authors examined cluster levels, growth rates, and mutant frequencies in Chinese hamster ovary cells expressing the Escherichia coli glycosylase fpg protein, whose principal substrates are oxidized purines. In cells expressing high levels of fpg protein, the levels of oxypurine clustered damages were decreased while those of oxypyrimidine clusters and abasic clusters were unchanged. Furthermore, in these cells, the growth rates were increased and the level of spontaneous background mutants in the hypoxanthine guanine phosphoribosyl transferase gene was decreased. These results suggest that endogenous clusters are potentially detrimental DNA damages, and that their levels-as well as the detrimental consequences of their presence-can be effectively reduced by increased cellular activity of specific DNA repair proteins.

Spectrum of complex DNA damages depends on the incident radiation.

Ionizing radiation induces bistranded clustered damages--two or more abasic sites, oxidized bases and strand breaks on opposite DNA strands within a few helical turns. Since clusters are refractory to repair and are potential sources of double-strand breaks (DSBs), they are potentially lethal and mutagenic. Although induction of single-strand breaks (SSBs) and isolated lesions has been studied extensively, little is known about the factors affecting induction of clusters other than DSBs. To determine whether the type of incident radiation could affect the yields or spectra of specific clusters, we irradiated genomic T7 DNA, a simple 40-kbp linear, blunt-ended molecule, with ion beams [iron (970 MeV/nucleon), carbon (293 MeV/nucleon), titanium (980 MeV/nucleon), silicon (586 MeV/nucleon), protons (1 GeV/nucleon)] or 100 kVp X rays and then quantified DSBs, Fpg-oxypurine clusters and Nfo-abasic clusters using gel electrophoresis, electronic imaging and number average length analysis. The yields (damages/Mbp Gy(-1)) of all damages decreased with increasing linear energy transfer (LET) of the radiation. The relative frequencies of DSBs compared to abasic and oxybase clusters were higher for the charged particles-including the high-energy, low-LET protons-than for the ionizing photons.

Endogenous DNA damage clusters in human skin, 3-D model, and cultured skin cells.

Clustered damages-two or more oxidized bases, abasic sites, or strand breaks on opposing DNA strands within a few helical turns-are formed in DNA by ionizing radiation. Clusters are difficult for cells to repair and thus pose significant challenges to genomic integrity. Although endogenous clusters were found in some permanent human cell lines, it was not known if clusters accumulated in human tissues or primary cells. Using high-sensitivity gel electrophoresis, electronic imaging, and number average length analysis, we determined endogenous cluster levels in DNA from human skin, a 3-D skin model, and primary cultured skin cells. DNA from dermis and epidermis of human skin contained extremely low levels of endogenous clusters (a few per gigabase). However, cultured skin fibroblasts and keratinocytes-whether in monolayer cultures or in 3-D model skin cultures-accumulated oxidized pyrimidine, oxidized purine, and abasic clusters. The levels of endogenous clusters were decreased by growing cells in the presence of selenium or by increasing cellular levels of Fpg protein, presumably by increasing processing of clustered damages. These results imply that the levels of endogenous clusters can be affected by the cells' external environment and their ability to deal with DNA damage.

Spontaneously occurring mutations in the cyclobutane pyrimidine dimer photolyase gene cause different sensitivities to ultraviolet-B in rice.

Sensitivity to ultraviolet-B (UVB) radiation (280-320 nm) varies widely among rice cultivars. We previously indicated that UV-resistant rice cultivars are better able to repair cyclobutane pyrimidine dimers (CPDs) through photorepair than are UV-sensitive cultivars. In this paper, we report that UVB sensitivity in rice, in part, is the result of defective CPD photolyase alleles. Surjamkhi (indica) exhibited greater sensitivity to UVB radiation and was more deficient in CPD photorepair ability compared with UV-resistant Sasanishiki (japonica). The deficiency in CPD photorepair in Surjamkhi resulted from changes in two nucleotides at positions 377 and 888 in the photolyase gene, causing alterations of two deduced amino acids at positions 126 and 296 in the photolyase enzyme. A linkage analysis in populations derived from Surjamkhi and Sasanishiki showed that UVB sensitivity is a quantitative inherited trait and that the CPD photolyase locus is tightly linked with a quantitative trait locus that explains a major portion of the genetic variation for this trait. These results suggest that spontaneously occurring mutations in the CPD photolyase gene cause different degrees of sensitivity to UVB in rice, and that the resistance of rice to UVB radiation could be increased by increasing the photolyase function through conventional breeding or bioengineering.

Processing of bistranded abasic DNA clusters in gamma-irradiated human hematopoietic cells.

Clustered DNA damages--two or more lesions on opposing strands and within one or two helical turns--are formed in cells by ionizing radiation or radiomimetic antitumor drugs. They are hypothesized to be difficult to repair, and thus are critical biological damages. Since individual abasic sites can be cytotoxic or mutagenic, abasic DNA clusters are likely to have significant cellular impact. Using a novel approach for distinguishing abasic clusters that are very closely spaced (putrescine cleavage) or less closely spaced (Nfo protein cleavage), we measured induction and processing of abasic clusters in 28SC human monocytes that were exposed to ionizing radiation. gamma-rays induced approximately 1 double-strand break: 1.3 putrescine-detected abasic clusters: 0.8 Nfo-detected abasic clusters. After irradiation, the 28SC cells rejoined double-strand breaks efficiently within 24 h. In contrast, in these cells, the levels of abasic clusters decreased very slowly over 14 days to background levels. In vitro repair experiments that used 28SC cell extracts further support the idea of slow processing of specific, closely spaced abasic clusters. Although some clusters were removed by active cellular repair, a substantial number was apparently decreased by 'splitting' during DNA replication and subsequent cell division. The existence of abasic clusters in 28SC monocytes, several days after irradiation suggests that they constitute persistent damages that could lead to mutation or cell killing.

Are endogenous clustered DNA damages induced in human cells?

Although clustered DNA damages are induced in cells by ionizing radiation and can be induced artifactually during DNA isolation, it was not known if they are formed in unirradiated cells by normal oxidative metabolism. Using high-sensitivity methods of quantitative gel electrophoresis, electronic imaging, and number average length analysis, we found that two radiosensitive human cell lines (TK6 and WI-L2-NS) accumulated Fpg-oxidized purine clusters and Nth-oxidized pyrimidine clusters but not Nfo-abasic clusters. However, four repair-proficient human lines (MOLT 4, HL-60, WTK1, and 28SC) did not contain significant levels (<5/Gbp) of any cluster type. Cluster levels were independent of p53 status. Measurement of glycosylase levels in 28SC, TK6, and WI-L2-NS cells suggested that depressed hOGG1 and hNth activities in TK6 and WI-L2-NS could be related to oxybase cluster accumulation. Thus, individuals with DNA repair enzyme deficiencies could accumulate potentially cytotoxic and mutagenic clustered DNA damages. The absence of Nfo-detected endogenous clusters in any cells examined suggests that abasic clusters could be a signature of cellular ionizing radiation exposure.

Quantifying clustered DNA damage induction and repair by gel electrophoresis, electronic imaging and number average length analysis.

Assessing DNA damage induction, repair and consequences of such damages requires measurement of specific DNA lesions by methods that are independent of biological responses to such lesions. Lesions affecting one DNA strand (altered bases, abasic sites, single strand breaks (SSB)) as well as damages affecting both strands (clustered damages, double strand breaks) can be quantified by direct measurement of DNA using gel electrophoresis, gel imaging and number average length analysis. Damage frequencies as low as a few sites per gigabase pair (10(9)bp) can be quantified by this approach in about 50ng of non-radioactive DNA, and single molecule methods may allow such measurements in DNA from single cells. This review presents the theoretical basis, biochemical requirements and practical aspects of this approach, and shows examples of their applications in identification and quantitation of complex clustered damages.

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.

Evaluation of number average length analysis in quantifying double strand breaks in genomic DNAs.

Double strand breaks in DNA can be quantified down to very low frequencies (a few per Gigabase pair) in nanogram quantities of nonradioactive, genomic DNA by dispersing the DNAs on electrophoretic gels, digitizing them by quantitative electronic imaging, and calculating the DNA lengths by number average length analysis. No specific distribution of damages is required for number average length analysis. To test the validity of this approach, we used DNA populations of known absolute lengths and break frequencies as experimental DNAs and calculated the number average lengths and double strand break levels. Experimental DNAs and length standards were dispersed using pulsed field electrophoretic modes (unidirectional pulsed field, contour clamped homogeneous field, or transverse alternating field) appropriate for their size range, stained with ethidium, destained, and a quantitative electronic image obtained. A dispersion curve was constructed from the migration-mobility relationships of the length standard DNAs, and the number average lengths of the experimental DNAs were calculated. The calculated DNA lengths agreed well with the actual lengths. Furthermore, the double strand break frequencies calculated through number average length analysis of DNAs dispersed by these pulsed field gel modes and digitized by quantitative electronic imaging were in excellent agreement with the actual values for populations of DNA over the size range of approximately 4 kbp to approximately 3 Mbp. The use of this approach in quantifying DNA damages is illustrated for double strand breaks and damage clusters (e.g., OxyPurine clusters recognized by Escherichia coli Fpg protein) induced in T7 DNA by ionizing radiation.

Quantifying double-strand breaks and clustered damages in DNA by single-molecule laser fluorescence sizing.

Fluorescence from a single DNA molecule passing through a laser beam is proportional to the size (contour length) of the molecule, and molecules of different sizes can be counted with equal efficiencies. Single-molecule fluorescence can thus determine the average length of the molecules in a sample and hence the frequency of double-strand breaks induced by various treatments. Ionizing radiation-induced frank double-strand breaks can thus be quantified by single-molecule sizing. Moreover, multiple classes of clustered damages involving damaged bases and abasic sites, alone or in combination with frank single-strand breaks, can be quantified by converting them to double-strand breaks by chemical or enzymatic treatments. For a given size range of DNA molecules, single-molecule sizing is as or more sensitive than gel electrophoresis, and requires several orders-of-magnitude less DNA to determine damage levels.

Repair of cyclobutyl pyrimidine dimers in human skin: variability among normal humans in nucleotide excision and in photorepair.

BACKGROUND/AIMS: Photoreactivation (PR) of cyclobutyl pyrimidine dimers (CPD) in human skin remains controversial. Recently Whitmore et al. (1) reported negative results of experiments using two photorepair light (PRL) sources on UV-irradiated skin of volunteers. However, their PRL sources induced substantial levels of dimers in skin, suggesting that the additional dimers formed could have obscured PR. We met a similar problem of dimer induction by a PRL source. We designed and validated a PRL source of sufficient intensity to catalyse PR, but that did not induce CPD, and used it to measure photorepair in human skin. METHODS AND RESULTS: Using a solar simulator filtered with three types of UV-filters, we found significant dimer formation in skin, quantified by number average length analysis using electrophoretic gels of isolated skin DNA. To prevent scattered UV from reaching the skin, we interposed shields between the filters and skin, and showed that the UV-filtered/shielded solar simulator system did not induce damage in isolated DNA or in human skin. We exposed skin of seven healthy human volunteers to 302 nm radiation, then to the improved PRL source (control skin areas were kept in the dark for measurement of excision repair). CONCLUSIONS: Using a high intensity PRL source that did not induce dimers in skin, we found that three of seven subjects carried out rapid photorepair of dimers; two carried out moderate or slow dimer photorepair, and three did not show detectable photorepair. Excision repair was similarly variable in these volunteers. Subjects with slower excision repair showed rapid photorepair, whereas those with rapid excision generally showed little or no photoreactivation.