Multiplicity of DNA single-strand breaks produced in pUC18 exposed to the direct effects of ionizing radiation

The transition of plasmid DNA from a supercoiled to an open circle conformation, as detected by gel electrophoresis, affords an extraordinarily sensitive method for detecting single-strand breaks (SSBs), one measure of deoxyribose damage. To determine the yield of SSBs, G(ssb), by this method, it is commonly assumed that Poisson statistics apply such that, on average, one SSB occurs per supercoiled plasmid lost. For the direct effect, at a large enough plasmid size, this assumption may be invalid. In this report, the assumption that one SSB occurs per pUC18 plasmid (2686 bp) is tested by measuring free base release (fbr), which is also a measure of deoxyribose damage in films prepared under controlled relative humidity so as to produce known levels of DNA hydration. The level of DNA hydration, Gamma, is expressed in mol water/mol nucleotide. The yield of free base release, G(fbr), was measured by HPLC after exposure of the films to 70 kV X rays and subsequent dissolution in water. It is well known that damage in deoxyribose leads to SSBs and free base release. Based on known mechanisms, there exists a close correspondence between free base release and SSBs, i.e., G(fbr) congruent with G(ssb). Following this assumption, the SSB multiplicity, m(ssb), was determined, where m(ssb) was defined as the mean number of SSBs per supercoiled plasmid lost. The yield of lost supercoil was determined previously (S. Purkayastha et al., J. Phys. Chem. B 110, 26286-26291, 2006). We found that m(ssb) = 1.4 +/- 0.2 at Gamma = 2.5 and m(ssb) = 2.8 +/- 0.5 to 3.1 +/- 0.5 at Gamma = 22.5, indicating that the assumption of one SSB per lost supercoil is not likely to hold for a 2686-bp plasmid exposed to the direct effect. In addition, an increase in G(fbr), upon stepping from Gamma = 2.5 to Gamma = 22.5, was paralleled by an increase in the yield of trapped deoxyribose radicals, G(dRib)(fr), also measured previously. As a consequence, the shortfall between SSBs and trapped radicals, G(diff) = G(ssb) - G(dRib)(fr), remained relatively constant at 90-110 nmol/J. The lack of change between the two extremes of hydration is in keeping with the suggestion that non-radical species, such as doubly oxidized deoxyribose, are responsible for the shortfall.     

DNA damage in cells exposed to ionizing radiation

Ionizing radiation induces variety of structural lesions in DNA of irradiated organisms. Their formation depends largely on the degree of cell oxygenation, the level of endogenous antioxidants, on DNA-protein complexes and compactization of DNA in the chromatin and activity of DNA repair systems. All ionizing radiation-induced DNA lesions can arbitrarily be divided into two groups. Group 1 includes singly damaged sites (single-sites): base modification, single-strand breaks, alkaline-labile sites (including a basic sites). Group 2 contains: locally multiply damaged sites (clustered lesions), double-strand breaks, intermolecular cross-links. The yields of lesions of group 2 increases with high linear energy transfer of radiation and these lesions play a dominant role in the radiation death, formation of chromosome and gene mutations, cell transformation.     

Photoreactivation of damage induced by ionizing radiation in yeast cells

Summary Experimental data on photoreactivation of damage induced by ionizing radiation in yeast cells are presented. The value of photoreactivation was found to be the highest for the following conditions predicted by us as optimum ones: large volume of irradiated suspension, hypoxia and high energy sparsely ionizing radiation. A comparison of data for yeast and bacterial cells shows that Cerenkov emission from ionizing radiation may produce photoreactivated pyrimidine dimers in both prokaryotic and eukaryotic cell systems.     

Construction of mobilizable vectors derived from plasmids RP4, pUC18 and pUC19

Mobilizable narrow-host-range plasmids were constructed from pUC18 and pUC19 by addition of a segment of pSUP2021 bearing the basis of mobilization (bom) site and origin of transfer (oriT) of RP4. One pair of expression vectors, pARO180 and pARO190, retains the beta-lactamase (bla) gene and twelve of the 13 restriction enzyme multiple cloning sites (MCS) of pUC18/19. Another pair was created by replacing the bla gene with the gene encoding kanamycin resistance (kan) from Tn5. The molecules replicate to high copy number in Escherichia coli and Enterobacter aerogenes. They can be transferred efficiently to other Gram- bacteria from the mobilizing strain, E. coli S17-1. In non-enteric strains, the new plasmids can be used as suicide vectors in site-specific insertional mutagenesis.     

Characterization of condensed plasmid DNA models for studying the direct effect of ionizing radiation

We have examined the changes in physical properties of aqueous solutions of the plasmid pUC18 that take place on the addition of the cationic oligopeptide penta-arginine. An increase in sedimentation rate and static light scattering, and changes in the nucleic acid CD spectrum all suggest that this ligand acts to condense the plasmid. Dynamic light scattering suggests the hydrodynamic radii of the condensate particles are a few micrometers, ca. 50-fold larger than that of the monomeric plasmid. Condensation of the plasmid also produces a ca. 100-fold decrease in the strand break yield produced by gamma irradiation. This extensive protection against reactive intermediates in the bulk of the solution implies that condensed plasmid DNA may offer a model system with which to study the direct effect of ionizing radiation (ionization of the DNA itself). The use of peptide ligands as condensing agents in this application is attractive because the derivatives of several amino acids (particularly tryptophan and tyrosine) have been shown to modify the radiation chemistry of DNA extensively.     

Unaltered free base release from d(CGCGCG)2 produced by the direct effect of ionizing radiation at 4 K and room temperature

Unaltered free base release in d(CGCGCG)2 exposed to X rays at 4 K or room temperature was measured by HPLC. Samples were prepared either as films hydrated to a level of Gamma = 2.5 mol water/mol nucleotide or as polycrystalline with Gamma approximately 7.5 mol water/mol nucleotide. X irradiation of films at 4 K, followed by annealing to room temperature, resulted in yields for cytosine and guanine of G(Cyt) = 0.036 +/- 0.001 micromol/J and G(Gua) = 0.090 +/- 0.002 micromol/J. Irradiation of films at room temperature gave similar yields. The yields for polycrystalline d(CGCGCG)2 X-irradiated at room temperature were G(Cyt) = 0.035 +/- 0.005 micromol/J and G(Gua) = 0.077 +/- 0.023 micromol/J. The total free base release yield, G(fbr), was 0.124 +/- 0.008 micromol/J for films and 0.112 +/- 0.028 micromol/J for polycrystalline samples. G(fbr) is believed to be a good estimate of total strand break yield. The yields of total free radicals trapped [G(Sigmafr)] by the d(CGCGCG)2 films at 4 K were measured by EPR. The measured value, G(Sigmafr) = 0.450 +/- 0.005 micromol/J, was used to calculate the yield of trappable sugar radicals, giving G(sugar)(fr) = 0.04-0.07 micromol/J. We found that (1) guanine release exceeded cytosine release by more than twofold, (2) G(sugar)(fr) cannot account for more than half of the free base release, and (3) G(fbr), G(Cyt) and G(Gua) were independent of the sample temperature during irradiation. Finding (1) suggests that base and or sequence influences sugar damage, and finding (2) is consistent with our working hypothesis that an important pathway to strand break formation entails two one-electron oxidations at the same sugar site.     

Exposure of human lymphocytes to ionizing radiation reduces mutagenesis by subsequent ionizing radiation

The effect of prior incubation with [3H]thymidine on survival and mutagenesis after X-irradiation of human lymphocytes was studied by incubating lymphocytes with 0.001-1.0 mu Ci/ml [3H]thymidine for 6 h at 37 degrees C and then irradiating with 150 or 300 rad. Survival was measured using lymphocyte cloning and mutagenesis was measured using 6-thioguanine selection to detect clones mutated at the hypoxanthine phosphoribosyltransferase locus. [3H]Thymidine alone had no effect on survival or mutagenesis and X-radiation alone produced the expected decrease in survival and increase in mutations. [3H]Thymidine prior to X-radiation had no effect on lethality of X-radiation but at concentrations of 0.1 and 1.0 mu Ci/ml produced a significant decrease in the number of mutations induced after both 150 and 300 rad. The results suggest that ionizing radiation, produced by disintegration of 3H, reduces the mutagenic effect of a subsequent exposure to ionizing radiation by induction of a system which prevents or repairs a restricted class of radiation damage.     

Reparable and irreparable damage in yeast cells induced by sparsely ionizing radiation.

It is shown that in diploid yeast there are significant differences in the extent of irreparable damage after irradiation with X-rays, 60Co-gamma-rays and 30 MeV electrons. At extremely low dose rates, 60Co-gamma-rays were found to produce almost no irreparable damage at least up to 1200 Gy. X-rays, however, at the same low dose rate caused irreparable damage in the same dose range yielding a surviving fraction of 0.25 at 1200 Gy. For irradiations at high dose rate followed by liquid holding recovery the relative biological effectiveness of X-rays amounted to at least 4 for absorbed doses of up to 1000 Gy. With 30 MeV electrons at high dose rates an accumulation of sublethal and potentially lethal damage resulting in irreparable damage occurred above 1000 Gy. It is suggested that irreparable damage in yeast is due to a cooperative effect of neighbouring track ends.     

Mechanisms of direct radiation damage in DNA, based on a study of the yields of base damage, deoxyribose damage, and trapped radicals in d(GCACGCGTGC)(2)

Dose-response curves were measured for the formation of direct-type DNA products in X-irradiated d(GCACGCGTGC)(2)prepared as dry films and as crystalline powders. Damage to deoxyribose (dRib) was assessed by HPLC measurements of strand break products containing 3' or 5' terminal phosphate and free base release. Base damage was measured using GC/ MS after acid hydrolysis and trimethylsilylation. The yield of trappable radicals was measured at 4 K by EPR of films X-irradiated at 4 K. With exception of those used for EPR, all samples were X-irradiated at room temperature. There was no measurable difference between working under oxygen or under nitrogen. The chemical yields (in units of nmol/J) for trapped radicals, free base release, 8-oxoGua, 8-oxoAde, diHUra and diHThy were G(total)(fr) = 618 +/- 60, G(fbr) = 93 +/- 8, G(8-oxoGua) = 111 +/- 62, G(8-oxoAde) = 4 +/- 3, G(diHUra) = 127 +/- 160, and G(diHThy) = 39 +/- 60, respectively. The yields were determined and the dose-response curves explained by a mechanistic model consisting of three reaction pathways: (1) trappable-radical single-track, (2) trappable-radical multiple-track, and (3) molecular. If the base content is projected from the decamer's GC:AT ratio of 4:1 to a ratio of 1:1, the percentage of the total measured damage (349 nmol/J) would partition as follows: 20 +/- 16% 8-oxoGua, 3 +/- 3% 8-oxoAde, 28 +/- 46% diHThy, 23 +/- 32% diHUra, and 27 +/- 17% dRib damage. With a cautionary note regarding large standard deviations, the projected yield of total damage is higher in CG-rich DNA because C combined with G is more prone to damage than A combined with T, the ratio of base damage to deoxyribose damage is approximately 3:1, the yield of diHUra is comparable to the yield of diHThy, and the yield of 8-oxoAde is not negligible. While the quantity and quality of the data fall short of proving the hypothesized model, the model provides an explanation for the dose-response curves of the more prevalent end products and provides a means of measuring their chemical yields, i.e., their rate of formation at zero dose. Therefore, we believe that this comprehensive analytical approach, combined with the mechanistic model, will prove important in predicting risk due to exposure to low doses and low dose rates of ionizing radiation.     

The role of miRNA in the direct and indirect effects of ionizing radiation

This review focuses on a number of recent studies that have examined changes in microRNA (miRNA) expression profiles in response to ionizing radiation and other forms of oxidative stress. In both murine and human cells and tissues, a number of miRNAs display significant alterations in expression levels in response to both direct and indirect radiation exposure. In terms of direct irradiation, or exposure to agents that induce oxidative stress, miRNA array analyses indicate that a number of miRNAs are up- and down-regulated and, in particular, the let-7 family of miRNAs may well be critical in the cellular response to oxidative stress. In bystander cells that are not directly irradiated, but close to, or share media with directly irradiated cells or tissues, the miRNA expression profiles are also altered, but are somewhat distinct from the directly irradiated cells. Based on the results of these numerous studies, as well as our own data presented here, we conclude that miRNA regulation is a critical step in the cellular response to radiation and oxidative stress and that future studies should elucidate the mechanisms through which this altered regulation affects cell metabolism.     

Differential oxidation of deoxyribose in DNA by gamma and alpha-particle radiation

Emerging evidence points to the importance of deoxyribose oxidation in the toxicity of oxidative DNA damage, including the formation of protein-DNA crosslinks and base adducts. With the goal of understanding the differences in deoxyribose oxidation chemistry known to occur with different oxidants, we have compared the formation of one product of 3'-oxidation of deoxyribose in DNA, 3'-phosphoglycolaldehyde (PGA) residues, in isolated DNA and cells exposed to ionizing radiations. A recently developed gas chromatography/negative chemical ionization mass spectrometry method was used to quantify PGA residues in purified DNA and in human TK6 lymphoblastoid cells exposed to gamma radiation (60Co) and alpha particles (241Am). The level of PGA residues was then correlated with the total quantity of deoxyribose oxidation determined by plasmid topoisomer analysis. Alpha-particle irradiation (0-100 Gy) of purified DNA in 50 mM potassium phosphate (pH 7.4) produced a linear dose response of 0.13 PGA residues per 10(6) nucleotides per gray. When normalized to an estimate of the total number of deoxyribose oxidation events (2.0 per 10(6) nucleotides per gray), PGA formation occurred in 7% (+/-0.5) of deoxyribose oxidation events produced by alpha-particle radiation. In contrast, the efficiency of PGA formation in gamma-irradiated DNA was found to be 1% (+/-0.02), which indicates a shift in the chemistry of deoxyribose oxidation, possibly as a result of the different track structures of the two types of ionizing radiation. Studies with gamma radiation were extended to TK6 cells, in which it was observed that gamma radiation produced a linear dose response of 0.0019 PGA residues per 10(6) nucleotides per gray. This is consistent with an approximately 1000-fold quenching effect in cells, similar to the results of other published studies of oxidative DNA damage in vivo.     

Antioxidative effects of melatonin in protection against cellular damage caused by ionizing radiation

Ionizing radiation is classified as a potent carcinogen, and its injury to living cells is, to a large extent, due to oxidative stress. The molecule most often reported to be damaged by ionizing radiation is DNA. Hydroxyl radicals (*OH), considered the most damaging of all free radicals generated in organisms, are often responsible for DNA damage caused by ionizing radiation. Melatonin, N-acetyl-5-methoxytryptamine, is a well-known antioxidant that protects DNA, lipids, and proteins from free-radical damage. The indoleamine manifests its antioxidative properties by stimulating the activities of antioxidant enzymes and scavenging free radicals directly or indirectly. Among known antioxidants, melatonin is a highly effective scavenger of *OH. Melatonin is distributed ubiquitously in organisms and, as far as is known, in all cellular compartments, and it quickly passes through all biological membranes. The protective effects of melatonin against oxidative stress caused by ionizing radiation have been documented in in vitro and in vivo studies in different species and in in vitro experiments that used human tissues, as well as when melatonin was given to humans and then tissues collected and subjected to ionizing radiation. The radioprotective effects of melatonin against cellular damage caused by oxidative stress and its low toxicity make this molecule a potential supplement in the treatment or co-treatment in situations where the effects of ionizing radiation are to be minimized.     

Immunochemical determination of DNA damage induced by high doses of ionizing radiation

It was shown by the immunochemical method that DNA of X-irradiated E. coli cells of a radiosensitive mutant ABA88uvr A6 can react with the antiserum to thymine dimers which, in all appearance, are induced by ionizing radiation in bacterial DNA. The number of thymine dimers in DNA of E. coli AB1886uvr A6 increased with the dose increase. No dimers were detected in radioresistant cells of M. radioproteolyticus probably due to the effective excision thereof.