Steven G. Swarts

Radiation-induced DNA damage as a function of hydration. I: Release of unaltered bases

The release of unaltered bases from irradiated DNA, hydrated between 2.5 and 32.7 mol of water per mole of nucleotide (gamma), was investigated using HPLC. The objective of this study was to elucidate the yield of the four DNA bases as a function of dose, extent of hydration, and the presence or absence of oxygen. The increase in the yield of radiation-induced free bases was linear with dose up to 90 kGy, except for the DNA with gamma = 2.5, for which the increase was linear only to 10 kGy. The yield of free bases as a function of gamma was not constant in either the absence or the presence of oxygen over the range of hydration examined. For DNA with gamma between 2.5 and 15, the yield of free bases was nearly constant under nitrogen, but decreased under oxygen. However, for DNA with gamma greater than 15, the yield increased rapidly under both nitrogen and oxygen. The yield of free bases was described by a model that depended on two factors: 1) a change in the DNA conformation from a mixture of the A and C conformers in vacuum-dried DNA to predominantly the B conformer in the fully hydrated DNA, and 2) the proximity of the water molecules to the DNA. Irradiation of the inner water molecules (gamma less than 15) was less efficient than irradiation of the outer water molecules (gamma greater than 15), by a factor of approximately 3.3, in forming DNA lesions that resulted in the release of an unaltered base. This factor is similar to the previously published relative efficiency of 2.8 with which hydroxyl radicals and base cations induce DNA strand breaks. Our irradiation results are consistent with the hypothesis that the G value for the first 12-15 water molecules of the DNA hydration layer is the same as the G value for the form of DNA to which it is bound (i.e., the pseudo-C or the B form). Thus we suggest that the release of bases originating from irradiation of the hydration water is obtained predominantly: (1) by charge transfer from the direct ionization of the first 12-15 water molecules of the primary hydration layer and (2) by the attack of hydroxyl radicals generated in the outer, more loosely bound water molecules.

Antioxidant Properties of Quercetin

UNLABELLED Quercetin, a plant-derived aglycone form of flavonoid glycosides, has been used as a nutritional supplement and may be beneficial against a variety of diseases, including cancer. We examined the antioxidant properties of quercetin. The reduction potential of quercetin was measured at various pH values using voltammetric methods, and its total antioxidant capacity (TAC) was measured using the phosphomolybdenum method. The effect of quercetin on production of reactive oxygen species (ROS) and nitric oxide (NO) in LPS-stimulated human THP-1 acute monocytic leukemia cells was determined by flow cytometry using CM-H2DCFDA dye. The results were compared with curcumin, a natural product exhibiting a similar range of reported health benefits. RESULTS 1) Quercetin has a higher reduction potential compared with curcumin at three different pH settings and is comparable to Trolox at pH 7-9.5; 2) its TAC is 3.5 fold higher than curcumin; 3) it reduced LPS-induced ROS to near normal levels; 4) it reduced LPS-induced NO production. These data provide a physico-chemical basis for comparing antioxidants, with potential benefits individually or in combination.

Radiation-induced DNA damage as a function of hydration. II. Base damage from electron-loss centers

The induction of base damage products in gamma-irradiated DNA, hydrated between 2.5 and 32.8 moles of water per mole of nucleotide (tau), was investigated using the gas chromatography/mass spectrometry-selected ion monitoring technique. In general, the yields of the measured base damage products were found to be dependent on the extent of the hydration when the DNA was irradiated under nitrogen. At low hydrations (tau < or = 13), the highest yields of the measured products were found for 7,8-dihydro-8-oxo-guanine, 5,6-dihydrothymine and, to a lesser extent, 2,6-diamino-4-oxo-5-formamidopyrimidine, products which are consistent with the base radicals found in low-temperature ESR studies. At higher hydrations (tau < or = 13), changes in DNA conformation and an increase in the attack of bulk water radicals on DNA play a significant role in the formation of radiation-induced DNA base damage products. Additional findings in our study include: (1) the sum of the yields of the products formed from electron-loss centers is greater than the sum of the yields of the products formed from electron-gain centers, indicating that there might be other electron-gain products which have not been identified; (2) the combined yield for the base damage products and the release of unaltered bases at tau < or = 13 is constant, implying that radiation damage in the tightly bound water molecules of the primary hydration layer causes DNA damage (quasi-direct effect) that is similar to the damage caused by direct ionization of the DNA (direct effect); and (3) the yields of the individual base damage products that were formed from electron-loss centers can be modeled on the basis of both the known reactions that lead to the formation of the initial charged base radicals in irradiated DNA, and the known reactions that involve the conversion of these initial DNA radicals into their respective nonradical end products.

Clinical EPR: Unique Opportunities and Some Challenges

Electron paramagnetic resonance (EPR) spectroscopy has been well established as a viable technique for measurement of free radicals and oxygen in biological systems, from in vitro cellular systems to in vivo small animal models of disease. However, the use of EPR in human subjects in the clinical setting, although attractive for a variety of important applications such as oxygen measurement, is challenged with several factors including the need for instrumentation customized for human subjects, probe, and regulatory constraints. This article describes the rationale and development of the first clinical EPR systems for two important clinical applications, namely, measurement of tissue oxygen (oximetry) and radiation dose (dosimetry) in humans. The clinical spectrometers operate at 1.2 GHz frequency and use surface-loop resonators capable of providing topical measurements up to 1 cm depth in tissues. Tissue pO2 measurements can be carried out noninvasively and repeatedly after placement of an oxygen-sensitive paramagnetic material (currently India ink) at the site of interest. Our EPR dosimetry system is capable of measuring radiation-induced free radicals in the tooth of irradiated human subjects to determine the exposure dose. These developments offer potential opportunities for clinical dosimetry and oximetry, which include guiding therapy for individual patients with tumors or vascular disease by monitoring of tissue oxygenation. Further work is in progress to translate this unique technology to routine clinical practice.

Monte Carlo Simulations of Site-Specific Radical Attack to DNA Bases

Abstract Aydogan, B., Bolch, W. E., Swarts, S. G., Turner, J. E. and Marshall, D. T. Monte Carlo Simulations of Site-Specific Radical Attack to DNA Bases. Radiat. Res. 169, 223–231 (2008). An atomistic biophysical model permitting the calculation of initial attacks to a 38-bp representation of B-DNA base moieties by water radicals is presented. This model is based on a previous radiation damage model developed by Aydogan et al. (Radiat. Res. 157, 38–44, 2002). Absolute efficiencies for radical attack to the 38-bp DNA molecule are calculated to be 41, 0.8 and 15% for hydroxyl radical (·OH), hydrogen radical (H·), and hydrated electron (eaq), respectively. Among the nucleobases, guanine is found to have the highest percentage ·OH attack probability at 36%. Adenine, cytosine and thymine moieties have initial attack probabilities of 24, 18 and 22%, respectively. A systematic study is performed to investigate ·OH attack probabilities at each specified attack site in four molecular models including free bases, single nucleotides, single base pairs, and the central eight base pairs of the 38-bp DNA molecule. Cytosine is the free base moiety for which the closest agreement is observed between the model prediction and the experimental data. The initial ·OH attack probabilities for cytosine as the free base are calculated to be 72 and 28%, while experimental data are reported at 87 and 13% for the C5 and C6 positions on the base, respectively. In this study, we incorporated atomic charges to scale the site-specific ·OH reaction rates at the individual atomic positions on the pyrimidine and purine bases. Future updates to the RIDNA model will include the use of electron densities to scale the reaction rates. With respect to reactions of the aqueous electron with DNA, a comparison of the initial distribution of electron attack sites calculated in this study and experimental results suggests an extremely rapid and extensive redistribution of the e−aq after their initial reactions with DNA.

Site-Specific OH Attack to the Sugar Moiety of DNA: A Comparison of Experimental Data and Computational Simulation

Abstract Aydogan, B., Marshall, D. T., Swarts, S. G., Turner, J. E., Boone, A. J., Richards, N. G. and Bolch, W. E. Site-Specific OH Attack to the Sugar Moiety of DNA: A Comparison of Experimental Data and Computational Simulation. Radiat. Res. 157, 38–44 (2002). Little computational or experimental information is available on site-specific hydroxyl attack probabilities to DNA. In this study, an atomistic stochastic model of OH radical reactions with DNA was developed to compute relative OH attack probabilities at individual deoxyribose hydrogen atoms. A model of the self-complementary decamer duplex d(CCAACGTTGG) was created including Na+ counter ions and the water molecules of the first hydration layer. Additionally, a method for accounting for steric hindrance from nonreacting atoms was implemented. The model was then used to calculate OH attack probabilities at the various C-H sites of the sugar moiety. Results from this computational model show that OH radicals exhibit preferential attack at different deoxyribose hydrogens, as suggested by their corresponding percentage solvent-accessible surface areas. The percentage OH attack probabilities for the deoxyribose hydrogens [1H(5′)+2H(5′), H(4′), H(3′), 1H(2′)+2H(2′), H(1′)] were calculated as approximately 54.6%, 20.6%, 15.0%, 8.5% and 1.3%, respectively, averaged across the sequence. These results are in good agreement with the latest experimental site-specific DNA strand break data of Balasubramanian et al. [Proc. Natl. Acad. Sci. USA 95, 9738–9742 (1998)]. The data from this stochastic model suggest that steric hindrance from nonreacting atoms significantly influences site-specific hydroxyl radical attack probabilities in DNA. A number of previous DNA damage models have been based on the assumption that C(4′) is the preferred site, or perhaps the only site, for OH-mediated DNA damage. However, the results of the present study are in good agreement the experimental results of Balasubramanian et al. in which OH radicals exhibit preferential initial attack at sugar hydrogen atoms in the order 1H(5′)+2H(5′) > H(4′) > H(3′) > 1H(2′)+2H(2′) > H(1′).

Ex vivo analysis of irradiated fingernails: chemical yields and properties of radiation-induced and mechanically-induced radicals.

A qualitative and quantitative analysis of the radicals underlying the radiation-induced signal (RIS) in fingernails was conducted in an attempt to identify properties of these radicals that could be used for biodosimetry purposes. A qualitative analysis of RIS showed the presence of at least three components, two of which were observed at low doses (<50 Gy) and the third required higher doses (>500 Gy). The low dose signal, obtained by reconstruction, consists of a 10 gauss singlet at g = 2.0053 and an 18 gauss doublet centered at g = 2.0044. Based on the initial slope of the dose-response curve, the chemical (radical) yields of the radicals giving rise to the singlet and doublet were 327 (±113) and 122 (±9) nmol J−1 (standard error, SE), respectively. At doses below 50 Gy, the singlet signal is the dominant component. Above this dose range, the signal intensity of the singlet rapidly dose-saturates. At doses <50 Gy, there is a small contribution of the doublet signal that increases in its proportion of the RIS as dose increases. A third component was revealed at high dose with a spectral extent of ∼100 gauss and displayed peaks due to g anisotropy at g = 2.056, 2.026, and 1.996. The total radical yield calculated from the initial slope of the dose-response curve averaged 458 ± (116) nmol J−1 (SE) in irradiated nail clippings obtained from six volunteers. Such high yields indicate that nails are a strong candidate for biodosimetry at low doses. In a comparison of relative stabilities of the radicals underlying the singlet and doublet signals, the stability of the doublet signal is more sensitive to the moisture content of the nail than the singlet. This differential in radical stabilities could provide a method for removing the doublet signal under controlled exposures to high humidities (>70% relative humidity). The decay of the singlet signal in RIS varies with exposure of a nail clipping to differing ambient humidities. However, long exposures (>6 h) to relative humidities of 72–94% results in singlet intensities that approach 7.0 ± (3.2)% (standard deviation) of the original intensities in an irradiated nail. This result suggests the existence of a subpopulation of radicals underlying the singlet signal that is relatively insensitive to decay under exposure of nails even to high humidities. Therefore, exposures of an irradiated nail clipping under controlled humidities may provide a method for estimating the exposure dose of the nail that is based on the intensity of the signal of the humidity insensitive radical population underlying the singlet signal.

ELECTRON PARAMAGNETIC RESONANCE DOSIMETRY FOR A LARGE-SCALE RADIATION INCIDENT

Abstract With possibilities for radiation terrorism and intensified concerns about nuclear accidents since the recent Fukushima Daiichi event, the potential exposure of large numbers of individuals to radiation that could lead to acute clinical effects has become a major concern. For the medical community to cope with such an event and avoid overwhelming the medical care system, it is essential to identify not only individuals who have received clinically significant exposures and need medical intervention but also those who do not need treatment. The ability of electron paramagnetic resonance to measure radiation-induced paramagnetic species, which persist in certain tissues (e.g., teeth, fingernails, toenails, bone, and hair), has led to this technique becoming a prominent method for screening significantly exposed individuals. Although the technical requirements needed to develop this method for effective application in a radiation event are daunting, remarkable progress has been made. In collaboration with General Electric and through funding committed by the Biomedical Advanced Research and Development Authority, electron paramagnetic resonance tooth dosimetry of the upper incisors is being developed to become a Food and Drug Administration-approved and manufacturable device designed to carry out triage for a threshold dose of 2 Gy. Significant progress has also been made in the development of electron paramagnetic resonance nail dosimetry based on measurements of nails in situ under point-of-care conditions, and in the near future this may become a second field-ready technique. Based on recent progress in measurements of nail clippings, it is anticipated that this technique may be implementable at remotely located laboratories to provide additional information when the measurements of dose on-site need to be supplemented. The authors conclude that electron paramagnetic resonance dosimetry is likely to be a useful part of triage for a large-scale radiation incident.

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

Abstract Swarts, S. G., Gilbert, D. C., Sharma, K. K., Razskazovskiy, Y., Purkayastha, S., Naumenko, K. A. and Bernhard, W. A. 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. Radiat. Res. 168, 367–381 (2007). Dose–response curves were measured for the formation of direct-type DNA products in X-irradiated d(GCACGCGTGC)2prepared 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 Gtotal(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 decamers 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 ∼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.

Replication of Murine Mitochondrial DNA Following Irradiation

The effect of radiation on the mitochondrial genome in vivo is largely unknown. Though mitochondrial DNA (mtDNA) is vital for cellular survival and proliferation, it has little DNA repair machinery compared with nuclear DNA (nDNA). A better understanding of how radiation affects mtDNA should lead to new approaches for radiation protection. We have developed a new system using real-time PCR that sensitively detects the change in copy number of mtDNA compared with nDNA. In each sample, the DNA sequence coding 18S rRNA served as the nDNA reference in a run simultaneously with a mtDNA sequence. Small bowel collected 24 hours after 2 Gy or 4 Gy total body irradiation (TBI) exhibited increased levels of mtDNA compared with control mice. A 4 Gy dose produced a greater effect than 2 Gy. Similarly, in bone marrow collected 24 hours after 4 Gy or 7 Gy TBI, 7 Gy produced a greater response than 4 Gy. As a function of time, a greater effect was seen at 48 hours compared with 24 hours. In conclusion, we found that radiation increased the ratio of mtDNA:nDNA and that this effect seems to be tissue independent and seems to increase with radiation dose and duration following radiation exposure.