Balagurumoorthy, P., Chen, K., Adelstein, S. J. and Kassis, A. I. Auger Electron-Induced Double-Strand Breaks Depend on DNA Topology. Radiat. Res. 170, 70–82 (2008). From a structural perspective, the factors controlling and the mechanisms underlying the toxic effects of ionizing radiation remain elusive. We have studied the consequences of superhelical/torsional stress on the magnitude and mechanism of DSBs induced by low-energy, short-range, high-LET Auger electrons emitted by 125 I, targeted to plasmid DNA by m -[ 125 I]iodo- p -ethoxyHoechst 33342 ( 125 IEH). DSB yields per 125 I decay for torsionally relaxed nicked (relaxed circular) and linear DNA (1.74 ± 0.11 and 1.62 ± 0.07, respectively) are approximately threefold higher than that for torsionally strained supercoiled DNA (0.52 ± 0.02), despite the same affinity of all forms for 125 IEH. In the presence of DMSO, the DSB yield for the supercoiled form remains unchanged, whereas that for nicked and linear forms decreases to 1.05 ± 0.07 and 0.76 ± 0.03 per 125 I decay, respectively. DSBs in supercoiled DNA therefore result exclusively from direct mechanisms, and those in nicked and linear DNA, additionally, from hydroxyl radical-mediated indirect effects. Iodine-125 decays produce hydroxyl radicals along the tracks of Auger electrons in small isolated pockets around the decay site. We propose that relaxation of superhelical stress after radical attack could move a single-strand break lesion away from these pockets, thereby preventing further breaks in the complementary strand that could lead to DSBs.

Singh, A., Chen, K., Adelstein, S. J. and Kassis, A. I. Synthesis of Coumarin–Polyamine-Based Molecular Probe for the Detection of Hydroxyl Radicals Generated by Gamma Radiation. Radiat. Res. 168, 233–242 (2007). To develop a molecular probe for detection of hydroxyl radicals in the vicinity of DNA, the coumarin–polyamine complexes, N 1 , N 12 -bis[2-oxo-2 H -chromene-3-carbonyl]-1,12-diamine-4,9-diazadodecane ( 5 ) and tris[2-(2-oxo-2 H -chromene-3-carboxamido)ethyl]amine ( 7 ), and their hydroxylated derivatives, N 1 , N 12 -bis[7-hydroxy-2-oxo-2 H -chromene-3-carbonyl]-1,12-diamine-4,9-diazadodecane ( 6 ) and tris[2-(7-hydroxy-2-oxo-2 H -chromene-3-carboxamido)ethyl]amine ( 8 ), have been synthesized. Using computer-generated molecular modeling, the derivatives have been docked onto DNA dodecamer d(CGCGAATTCGCG) 2 , the ligand–DNA complexes have been minimized, and the free binding energies (Δ G binding ) and inhibition constants ( K i ) have been calculated. Compound 7 is not water-soluble at the concentrations required for the project. When aqueous solutions of 5 are irradiated with γ rays, the relationship between induced fluorescence and dose is linear in the range of 0 to 10 Gy. The fluorescence emission spectrum of irradiated 5 is similar to that of its dihydroxy derivative 6, indicating conversion of 5 to 6, and induction of fluorescence records formation of hydroxyl radicals in aqueous solution. The dicoumarin–polyamine 5, a novel compound for the detection of hydroxyl radicals close to DNA, is a sensitive and quantitative probe with potential for applications in biological systems.

Yasui, L. S., Chen, K., Wang, K., Jones, T. P., Caldwell, J., Guse, D. and Kassis, A. I. Using Hoechst 33342 to Target Radioactivity to the Cell Nucleus. Radiat. Res. 167, 167–175 (2007). We have explored the use of Hoechst 33342 (H33342) to carry radioactivity to the cell nucleus. H33342 enters cells and targets DNA at adenine-thymine-rich regions of the minor groove. Considerable membrane blebbing and ruffling occur in CHO cells within minutes after its addition to the culture medium in micromolar quantities. Blue vesicles are apparent in the cell cytoplasm, and by 30 min the nuclei are stained dark blue. Upon its binding to DNA, a visible emission shift of the dye can be observed with fluorescence microscopy. We have radioiodinated ( 125 I) H33342 and specifically irradiated nuclear DNA by incubating CHO cells with 125 I-H33342 at 37°C and accumulating 125 I decays at −90°C. At various times, the cells are thawed and assayed for survival (clonogenicity) and DSB (γ-H2AX) formation. 125 I-H33342 decay leads to a monoexponential decrease in cell survival with a D 0 of 122 125 I decays per cell and a linear increase in DNA DSB induction (equivalent to 15 γ-H2AX foci/cell). Cell death is not modified by the radioprotective effects of H33342 because we use considerably lower concentrations than those that provide a slight protection against γ radiation. We conclude that cell killing by 125 I-H33342 and the induction of γ-H2AX foci are highly correlated.

Balagurumoorthy, P., Chen, K., Bash, R. C., Adelstein, S. J. and Kassis, A. I. Mechanisms Underlying Production of Double-Strand Breaks in Plasmid DNA after Decay of 125 I-Hoechst. Radiat. Res. 166, 333–344 (2006). Previously, the kinetics of strand break production by 125 I-labeled m -iodo- p -ethoxyHoechst 33342 ( 125 IEH) in supercoiled (SC) plasmid DNA had demonstrated that ∼1 DSB is produced per 125 I decay both in the presence and absence of the hydroxyl radical scavenger DMSO. In these experiments, an 125 IEH:DNA molar ratio of 42:1 was used. We now hypothesize that this DSB yield (but not the SSB yield) may be an overestimate due to subsequent decays occurring in any of the 41 125 IEH molecules still bound to nicked (N) DNA. To test our hypothesis, 125 IEH was incubated with SC pUC19 plasmids ( 125 IEH:DNA ratio of ∼3:1) and the SSB and DSB yields were quantified after the decay of 125 I. As predicted, the number of DSBs produced per 125 I decay is one-half that reported previously (∼0.5 compared to ∼1, ± DMSO) whereas the number of SSBs (∼3/ 125 I decay) is similar to that obtained previously (∼90% are generated by OH radicals). Direct visualization by atomic force microscopy confirms formation of L and N DNA after 125 IEH decays in SC DNA and supports the strand break yields reported. These findings indicate that although SSB production is independent of the number of 125 IEH bound to DNA, the DSB yield can be augmented erroneously by 125 I decays occurring in N DNA. Further analysis indicates that 17% of SSBs and 100% of DSBs take place within the plasmid molecule in which an 125 IEH molecule decays, whereas 83% of SSBs are formed in neighboring plasmid DNA molecules.

Kishikawa, H., Wang, K., Adelstein, S. J. and Kassis, A. I. Inhibitory and Stimulatory Bystander Effects are Differentially Induced by Iodine-125 and Iodine-123. Radiat. Res. 165, 688–694 (2006). The bystander effect, originating from cells irradiated in vitro , describes responses of surrounding cells not targeted by the radiation. Previously we demonstrated that the subcutaneous injection into nude mice of human adenocarcinoma LS174T cells lethally irradiated by Auger electrons from the decay of DNA-incorporated 125 I inhibits growth of co-injected LS174T cells (inhibitory bystander effect; Proc. Natl. Acad. Sci. USA 99, 13765–13770, 2002). We have repeated these studies using cells exposed to lethal doses of 123 I, an Auger electron emitter whose emission spectrum is identical to that of 125 I, and report herein that the decay of 123 I within tumor cell DNA stimulates the proliferation of neighboring unlabeled tumor cells growing subcutaneously in nude mice (stimulatory bystander effect). Similar inhibitory bystander effects ( 125 I) and stimulatory bystander effects ( 123 I) are obtained in vitro . Moreover, supernatants from cultures with 125 I-labeled cells are positive for tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), and those from cultures with 123 I-labeled cells are positive for angiogenin. These findings call for the re-evaluation of current dosimetric approaches for the estimation of dose–response relationships in individuals after radiopharmaceutical administration or radiocontamination and demonstrate a need to adjust all “calculated” dose estimates by a dose modification factor (DMF), a radionuclide-specific constant that factors in hitherto not-so-well recognized biophysical processes.

To elucidate the nature and kinetics of DNA strand breaks caused by low-energy Auger electron emitters, we compared the yields of DNA breaks in supercoiled pUC19 DNA in the presence of the <tex-math>${}^{\bullet}{\rm OH}$</tex-math> scavenger dimethyl sulfoxide (DMSO) after the decay of 125 I (1) in proximity to DNA after minorgroove binding (<tex-math>${}^{125}{\rm I}\text{-iodoHoechst}$</tex-math> 33342, <tex-math>${}^{125}{\rm IH}$</tex-math>) and (2) at a distance from DNA (<tex-math>${}^{125}{\rm I}\text{-iodoantipyrine}$</tex-math>, <tex-math>${}^{125}{\rm IAP}$</tex-math>). DMSO is efficient at protecting supercoiled plasmid DNA from the decay of 125 I free in solution (dose modification factor, DMF = 59 ± 4) and less effective when the 125 I decays occur close to DNA (DMF = 3.8 ± 0.3). This difference is due mainly to the inability of DMSO to protect DNA from the double-strand breaks produced by groove-bound 125 I (DMF = 1.0 ± 0.2). Additionally, the fragmentation of plasmid DNA beyond the production of single-strand and double-strand breaks that is seen after the decay of <tex-math>${}^{125}{\rm IH}$</tex-math> and not <tex-math>${}^{125}{\rm IAP}$</tex-math> (Kassis et al., Radiat. Res. 151, 167-176, 1999) cannot be modified by DMSO. These results demonstrate that the mechanisms underlying double-strand breaks caused by the decay of <tex-math>${}^{125}{\rm IH}$</tex-math> differ in nature from those caused by the decay of <tex-math>${}^{125}{\rm IAP}$</tex-math>.

To elucidate the kinetics of the induction of DNA strand breaks by low-energy Auger electron emitters, we compared the yields of DNA breaks in supercoiled pUC19 DNA after the decay of 125 I (1) in proximity to DNA after minor-groove binding ( ${}^{125}{\rm I}\text{-iodoHoechst}$ 33342, ${}^{125}{\rm IH}$ ) and (2) at a distance from DNA ( ${}^{125}{\rm I}\text{-iodoantipyrine}$ , ${}^{125}{\rm IAP}$ ). Iodine-125 bound to the minor groove in DNA or free in solution is equally effective per decay in producing single-strand breaks (SSBs), while 125 I bound to the minor groove is 6.7-fold more efficient than 125 I free in solution in producing double-strand breaks (DSBs) (1.08 ± 0.13 compared to 0.16 ± 0.01 DSB/decay). Consequently, SSB to DSB ratios for ${}^{125}{\rm IAP}$ and γ radiation (20.7 ± 2.9 and 43.8 ± 1.5, respectively) are greater than that for ${}^{125}{\rm IH}$ (2.9 ± 0.4). Finally, the decay of ${}^{125}{\rm IH}$ leads to fragmentation of plasmid DNA beyond SSBs and DSBs.

Asynchronous Chinese hamster V79 lung fibroblasts were incubated at 37°C for 30 min with the thymidine analog $5\text{-}[{}^{211}{\rm At}]\text{astato-}2^{\prime}\text{-deoxyuridine}$ ( ${}^{211}{\rm AtdU}$ , exposure from DNA-incorporated activity) or with $[{}^{211}{\rm At}]\text{astatide}$ ( ${}^{211}{\rm At}$ , exposure from extracellular activity), and DNA-incorporated activity was determined. The ${}^{211}{\rm AtdU}$ content in cellular DNA increased as a function of extracellular concentration. Incorporation of ${}^{211}{\rm At}$ was less than 1% of that of ${}^{211}{\rm AtdU}$ . After exposure, cells were frozen in the presence of 10% DMSO. One month later, survival was determined by the colony-forming assay, and DNA double-strand breaks (DSBs) were measured by the neutral elution method (pH 9.6). The survival curve for ${}^{211}{\rm AtdU}$ was biphasic ( $D_{37}=2.8$ decays per cell), reflecting killing of ${}^{211}{\rm At}\text{-}{\rm DNA}\text{-labeled}$ cells and of unlabeled cells irradiated by ${}^{211}{\rm At}$ in neighboring labeled cells. The toxicity of ${}^{211}{\rm At}$ decaying outside the cell (30-min exposure) was negligible. Analysis of the survival curve produced a D 0 of 1.3 decays/cell for ${}^{211}{\rm At}\text{-labeled}$ cells. The yield of DSBs from the decay of DNA-incorporated ${}^{211}{\rm At}$ was compared with that from DNA-incorporated 125 I. Each decay of ${}^{211}{\rm At}$ produced at least 10 times the number of DSBs as that obtained per 125 I decay. The extreme radiotoxicity of DNA-incorporated ${}^{211}{\rm AtdU}$ seems to be associated with considerable damage to the mammalian cell genome.

We have examined whether mammalian cells in vitro can be protected against the lethal effects of irradiation by Auger electrons emitted from DNA-incorporated 125 I. Chinese hamster V79 lung fibroblasts were cultivated in the presence of <tex-math>$5\text{-}[{}^{125}{\rm I}]{\rm iodo}\text{-}2^{\prime}\text{-deoxyuridine}$</tex-math> (<tex-math>${}^{125}{\rm IdU}$</tex-math>) for 18 h and resuspended in ice-cold medium in the presence or absence of 10% dimethyl sulfoxide (DMSO). DNA-incorporated 125 I activity was measured and the cells were plated for survival. A portion of the cell suspensions were also stored on ice to accumulate 125 I decays for 6 to 48 h, after which the cells were plated to determine survival. Storage on ice up to 48 h without radioactivity reduced plating efficiency from 67 ± 4% (SEM) to 20 ± 1%. DMSO had a protective effect on colony formation, as the respective cloning efficiencies were 83 ± 3% and 72 ± 12% at 0 and 48 h. The survival curves for <tex-math>${}^{125}{\rm IdU}\text{-labeled}$</tex-math> cells are exponential with <tex-math>$D_{0}=36\pm 2$</tex-math> decays per cell in the absence of DMSO and 195 ± 20 decays per cell in the presence of DMSO. Thus the dose modification factor (DMF) at 37% survival for 10% DMSO is 5.4 ± 0.6 for DNA-incorporated 125 I. In reference experiments, a DMF of 2.5 ± 0.8 was measured for cells irradiated with 137 Cs γ rays. These results indicate that the radiotoxicity of Auger electrons from 125 I decay in mammalian cells is caused mainly by an indirect mechanism(s).

We have examined whether nuclear DNA can be protected from double-strand breaks (DSBs) induced by decay of the Auger-electron-emitting radionuclide 125 I. Decays were accumulated at 0.3°C in Chinese hamster V79 cells suspended in isotonic buffer containing 0.1 M EDTA in the presence or absence of 10% dimethyl sulfoxide (DMSO). DSBs were measured by the neutral elution method (pH 9.6) and quantified as strand scission factors. DMSO was shown to protect DNA from DSBs caused by the decay of DNA-incorporated 125 I. The dose modification factor (DMF) for this radionuclide decreases as a function of 125 I decays (389 to 4,100 decays, DMF = 2.5 to 1.3). Extrapolation of the curve for the DMF indicates that at ∼ 15,000 decays/cell, a DMF of 1 would be obtained. Experiments using large numbers of 125 I decays confirmed these extrapolations. For induction of DSBs by 137 Cs γ rays, the DMF also decreases with dose (50 to 290 Gy, DMF = 2.7 to 1.5). However, extrapolation of the curve for the DMF indicates that protection does not cease at higher doses. The data show that, at the same level of damage, DMSO can protect against γ-ray-induced DSBs 1.35-fold more efficiently than against DSBs caused by the decay of DNA-incorporated 125 I. It appears that when 125 I is incorporated into DNA, chromatin structure fosters some DSB formation by an indirect mechanism(s) and that more than one DSB is generated per decaying atom.

The chemotoxicity and radiotoxicity of trans-dichlorodiammineplatinum (II) labeled with ${}^{195{\rm m}}{\rm Pt}$ ( $trans\text{-}{}^{195{\rm m}}{\rm Pt}$ ) are investigated to ascertain the potential of radioplatinum coordination complexes as antineoplastic agents. Platinum-195m, with a half-life of about 4 days, is a prolific emitter of low-energy Auger electrons because of the high probability of internal conversion in its isomeric transitions. The kinetics of cellular uptake and retention after incubation and the radiotoxicity of this Auger electron emitter in the form of $trans\text{-}{}^{195{\rm m}}{\rm Pt}$ is investigated using cells of the Chinese hamster V79 cell line. The cellular uptake of ${}^{195{\rm m}}{\rm Pt}$ reaches a plateau in about 3 to 5 h of incubation and varies nonlinearly with the extracellular concentration of radioactivity. The radioactivity is eliminated from the cells after incubation with an effective half-life of 24 h. Cell survival data, when corrected for the chemical toxicity of nonradiolabeled trans-platinum, give a cell survival curve typical for radiations with high linear energy transfer. At 37% survival, the mean lethal cellular uptake is about 1.0 mBq/cell. Dosimetric considerations, based on subcellular distribution of the radionuclide, yield a value of 4.8 for the relative biological effectiveness when compared with 250 kVp X rays. Theoretical Monte Carlo track-structure calculations indicate that the density of radical species produced in liquid water in the immediate vicinity of a ${}^{195{\rm m}}{\rm Pt}$ decay site is substantially greater than the density of species along the track of a 5.3 MeV α particle. This explains qualitatively the efficacy of ${}^{195{\rm m}}{\rm Pt}$ in causing high-LET radiation type biological effects. The extreme radiotoxicity of intranuclearly localized ${}^{195{\rm m}}{\rm Pt}$ , in conjunction with the proclivity of platinum chemotherapy agents to bind to DNA in the cell nucleus, suggests that the combination of chemical effects and the effects of Auger electrons that can be obtained with radioplatinum coordination complexes may have potential in the treatment of cancer.

The response of cultured bovine aortic endothelial (BAE) cells after exposure to α-particle radiation from chelated 212 Bi has been evaluated. The results suggest that even relatively high doses of α-particle radiation from 212 Bi (20-72 Gy) cause only minor acute changes in the morphology of BAE cells (light and electron microscopy) under conditions of confluent monolayer growth. Significant morphological changes can be detected in cells that detach from the monolayer, though it is unclear whether these changes represent a genuine response to irradiation or reflect the causes or effects of monolayer detachment with the consequent loss of intercellular biochemical communication. After α-particle irradiation (20-40 Gy) angiotensin-converting-enzyme activity was not detectable in the monolayer culture medium but was significantly decreased within the cell monolayer. Neutral-elution-assay data demonstrated that DNA double-strand-break (DSB) damage occurred in these cells and that about 35% of the DSBs were repairable.

To explore the effect of the Auger electron emitter 125 I attached to a DNA intercalator, we have synthesized 125 I- and 127 I-labeled 3-acetamido-5-iodoproflavine (AIP) and have examined the uptake, intracellular distribution, and radiotoxicity of A 125 IP in Chinese hamster V79 cells. After incubation with AIP, the nuclei of V79 cells become fluorescent. Uptake of A 125 IP is directly proportional to its extracellular radioactive concentration and reaches a plateau at about 10 h. Of the cell-associated radioactivity, 60% is retained by the cells after extensive washing. When the survival of V79 cells is plotted as a function of radioactive cell content, the curve has no shoulder with a mean lethal dose ( D N ) of about 1.3 Gy to the cell nucleus. Because the D N of these cells when irradiated with 250 kVp X rays is 5.8 Gy, the relative biological effectiveness (RBE) of A 125 IP is about 4.5. The dependence of the RBE values on the localization of the Auger emitter is discussed on the basis of our extended studies on the same cell line.